Enteral feeding devices and related methods of use

ABSTRACT

Embodiments of the disclosure are drawn to an enteral feeding device for hydrolyzing triglycerides in a nutritional formula. The device may include a body housing a chamber, an inlet configured to fluidly couple with a source of nutritional formula, and an outlet configured to fluidly couple with an enteral feeding tube. The device may include a headspace and a plurality of particles contained within the chamber, wherein the lipase is covalently bonded to the plurality of particles. The device may include an inlet filter located between the inlet and the chamber, wherein the inlet filter contains a first plurality of openings, and an outlet filter located between the chamber and the outlet, wherein the outlet filter has a second plurality of openings smaller than the plurality of particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefits of priority from U.S. ProvisionalApplication No. 62/241,608, filed on Oct. 14, 2015, the entirety ofwhich is incorporated herein by reference.

FIELD OF THE DISCLOSURE

Embodiments of the present disclosure are directed to devices andmethods for processing a nutritional formula, and more particularly, todevices and methods for hydrolyzing fats in a nutritional formula intofree fatty acids and monoglycerides for ingestion.

BACKGROUND OF THE DISCLOSURE

Long-chain polyunsaturated fatty acids (LC-PUFAs) are lipids havinghydrocarbon chains containing two or more carbon-carbon double bonds.LC-PUFAs, such as docosahexaenoic acid (DHA), eicosapentaenoic acid(EPA), and arachidonic acid (AA), are critical for normal human growth,development, and maintaining caloric intake, have important visual,cognitive, cardiovascular, and immunological health benefits throughouta person's life and in medical treatments, and are important formaintaining and/or gaining weight and subsequent survival after medicaltreatments. The principal source for DHA and EPA is through diet and, toa lesser degree, their precursor, alpha-linolenic acid (ALA), an omega-3fatty acid. The principal source for AA is through the diet and, to alesser degree, linoleic acid (LA), an omega-6 fatty acid. Endogenouslyproduced enzymes are highly inefficient at converting ALA to DHA andEPA. According to an official statement by the International Society forthe Study of Fatty Acids and Lipids (ISSFAL), the conversion of ALA toDHA is about 1% in infants and is considerably lower in adults. Brennaet al., Prostaglandins Leukot Essent Fatty Acids, 80(2-3):85-91 (2009).Thus, adequate absorption of dietary and supplemental nutrient sourcesof LC-PUFAs, such as DHA and EPA, is important for the health of thehuman body. Until 2001, direct sources of DHA and AA were not part ofthe ingredients used in infant formulas in the US.

LC-PUFAs, such as DHA, EPA, and AA, in the diet are primarily in theform of long-chain triglycerides and/or long-chain fatty acid esters.Long-chain polyunsaturated triglycerides are made of three long-chainfatty acids bound to a glycerol molecule via ester linkages. Absorptionof long-chain triglycerides by the body first requires the enzymaticaction of lipase, e.g., pancreatic lipase, which digest triglyceridesthrough hydrolysis, breaking them down into monoglycerides and freefatty acids. As used herein, the terms triglycerides and fatty acidsboth may refer to fats found in food or supplemental nutritionalformulas. Fatty acids and monoglycerides are found as triglycerides insupplemental nutritional formulas. Free fatty acids or fatty acids notattached to other molecules are used to refer to the byproduct of fatdigestion. Free fatty acids or fatty acids not attached to othermolecules are unstable, which makes them unsuitable to be packaged insupplemental nutritional formulas.

Additionally, the chain lengths and the number of carbon-carbon doublebonds of fatty acids may influence fat absorption. Dietary fatty acidsfound in food are long-chain fatty acids having at least 12 carbons, forexample 16, 18, or 20 carbons, known as C16, C18, and C20 long-chainfatty acids. Medium-chain fatty acids having less than or equal to 12carbons, for example, 8 and 12 carbons, known as C8 and C12 are rarelyfound in food (except for coconuts) and are thus less important fordigestion and absorption in humans. Short-chain fatty acids having lessthan or equal to a few carbons, for example, 2, 3, and 4 carbons, knownas C2, C3, and C4, are the major anions found in the stool, but they arenot found in food. Short-chain fatty acids result from the digestion offats by the bacteria in the colon and thus often contribute to diarrheaby providing an osmotic gradient. B. Goodman, Adv. Physiol. Educ.,34(2):44-53 (2010).

While all fats provide caloric benefit, they have different impacts onphysiological functions. St-Ogne et al., J. Nutr., 132(3): 329-333(2002). Short-chain triglycerides and medium-chain triglycerides (MCTs)are absorbed directly through the villi of the intestinal mucosa. MCTscan be readily absorbed due to their shorter chain lengths and theresidual activity of gastric lipase, even in patients having compromisedpancreatic output or pancreatic insufficiency. Long-chain triglycerides(LCTs) have fatty acids with more than 12 carbons, for example C13 toC24. LCTs are not directly absorbed but instead must first be hydrolyzedinto free fatty acids and monoglycerides by pancreatic lipase beforethey are absorbed in the small intestine. Once free fatty acids andmonoglycerides are absorbed, they are transported to the liver andultimately to tissues in the body for various physiological purposes.While both LCTs and MCTs provide calories, only LCTs, specificallyLCPUFAs, provide structural components of membranes and biologicalmediators involved in the regulation of many physiological functions.MCTs, when substituted for LCTs, have been shown to increase energyexpenditure and satiety, leading to reduced overall caloric intake andreduced body fat mass. This makes MCTs a poor long-term energy sourcefor patients having compromised pancreatic output or pancreaticinsufficiency. M. Clegg, Int. J. Food Sci. Nutr., 61(7):653-79 (2010).Furthermore, DHA and EPA are commercially available as triglycerides orin esterified form in nutritional supplements, prescription products(e.g., LOVAZA®, OMACOR®, and Vascepa™), and infant formulas. Thesenutritional supplements or products may be in the form of a powder,liquid beverage, or enteral-feeding formula. Because polyunsaturatedfatty acids are unstable and can rapidly degrade, no enteral formula ornutritional supplements containing hydrolyzed fatty acids has beenmanufactured to date.

Some people, however, are unable to adequately break down or absorblong-chain triglycerides, structured fats, and/or long-chain esters,e.g., patients suffering from compromised pancreatic output orpancreatic insufficiency, pre-term infants, people in the ICU, and theelderly, and as a result, they may suffer from inadequate hydrolysis orabsorption of long-chain triglycerides and/or long-chain esters and maynot benefit from the intake of dietary and/or nutrient supplementsources of LC-PUFAs. Uncorrected fat malabsorption due to compromisedpancreatic and/or gastrointestinal or liver dysfunction can lead tomalnutrition, failure to gain or maintain weight, decreased ability torecover from infections, decreased growth, and impaired absorptivecapacity of the gastrointestinal lumen, despite adequate or exaggeratedfood intake.

For example, exocrine pancreatic insufficiency (EPI) is one of theconditions that lead to a reduced ability to hydrolyze long-chaintriglycerides. EPI may result from diseases that affect and destroy theexocrine function of the pancreas, including cystic fibrosis (CF),chronic pancreatitis (CP), surgery, cancer (in particular pancreatic),developmental immaturity, and pancreatectomy for the treatment of injuryor infection. In the course of EPI, lipid malabsorption with resultingsteatorrhea typically develops earlier than does the maldigestion ofproteins or carbohydrates. Weight loss and steatorrhea are common to allcancers due to the catabolic state of tissues, diversion of nutrients,and malabsorption in advanced stages. Pancreatic cancer is uniquecompared to other cancers, as weight loss and malabsorption are presentin 80%-90% of patients at the time of diagnosis. The vast majority ofpeople with EPI, including CF patients, have significantgastrointestinal manifestations (˜90%), leading to fatty acidalterations, imbalances and deficiencies of long-chain fatty acids,e.g., DHA and/or EPA, which may also contribute to the inflammatorycharacteristics of CF lung disease, such as chronic suppurative lungdisease and GI symptoms. In general, EPI may result in decreasedpancreatic lipase secretion or efficacy and maldigestion andmalabsorption of lipids, leading to reduced caloric intake, significantweight loss, LC-PUFA deficiencies, and GI symptoms, includingsteatorrhea with bulky, greasy, foul-smelling stools, pain, flatulence,nausea, and thus can have a significant impact on the quality of life.

Current options for treating EPI or to improve the absorption of dietaryor supplemental LC-PUFA intake, such as DHA and EPA, include addinglipase supplements to the diet or nutrient supplements to improvehydrolysis of long-chain triglycerides, including pancreatic lipase.However, pancreatic enzymes, and particularly pancreatic lipase presentin these supplements, are often sensitive to degradation by gastric acidand pepsin so that only a small fraction of the ingested enzymes reachthe duodenum in active form. E. Ville et al., Digestion, 65:73-81(2001). Further, most commercial lipase supplements are made from animalpancreatic lipase, which is known to have significantly reducedstability below a pH of 7. See, e.g., US2010/0239559; D. Kasper et al.,Harrison's Principles of Internal Medicine 16^(th) Ed. (2004). By thetime such lipases pass through the stomach, significant amounts arelikely to have been inactivated. Also, not all lipases work to the samedegree for hydrolysis of a given long-chain fatty acid, indicatinglipase specificity is an important consideration. R. Jensen et al.,Lipids, 18(3):239-252 (1983). And, in some populations with EPI,nutritional formulas are tightly regulated, such as in pre-term infantsor in patients in intensive care units. For these controlledpopulations, it may not be desirable or feasible to supplementalready-approved formulas with additional ingredients.

The current standard of care for treating fat malabsorption andimproving dietary fat intake includes porcine enzymatic replacementtherapy (PERT) and the use of exaggerated levels of fats delivered asMCTs. In PERT, porcine-derived pancreatic enzyme products areadministered orally with meals and snacks. The porcine-derivedpancreatic enzymes are typically extracted from pancreas glandsharvested from pigs used for food consumption in slaughterhousescertified by the US Department of Agriculture or comparable Europeanauthorities. These porcine-derived pancreatic enzymes may contain amixture of enzymes including lipases, trypsin, chymotrypsin, elastase,proteases, and amylases, and other cellular components. The use andreliance on porcine-sourced material in these products may posepotential risks, including human infection with zoonotic viruses,exposure to endogenous porcine viruses, allergic reactions, and thepresentation of hyperuricemia. Moreover, the availability ofporcine-derived pancreatic enzymes can be a concern in the event thatsource herds need to be culled due to diseases or other agriculturalimperatives.

Furthermore, lipase supplements, such as the porcine-derived pancreaticenzymes, must be covered with a polymeric resin coating(hydroxypropyl-methylcellulose phthalate or other phthalates) to preventthem from being inactivated in the low-pH environment of the stomach.The polymeric coating approximately constitutes about 30% of the weightof such capsules and is non-digestible, absorbed systemically andexcreted by the kidneys. For these reasons, the use of PERTs in immunecompromised patients or infants, especially preterm infants, is notpractical due to the many potential safety concerns. Moreover, althoughacid protective coatings have helped, some degree of malabsorptionpersists, causing patients with EPI to require increasing doses ofenzyme supplements. This persistence of fatty acid malabsorption evenwith use of enterically coated enzymes may be due to the fact that theduodenum and upper jejunum in patients with EPI are often acidicenvironments, so that the expected raise in pH is not achieved, and theprotective coating is not properly dissolved to release the enzyme. D.Graham, New England J. Med., 296(23):1314-1317 (1977). Both of theseproblems have been addressed by increasing the dose of enzymesadministered. It has been observed that large amounts of pancreaticdigestive enzymes can damage the large intestine resulting in fibrosingcolonopathy. D. Bansi et al., Gut, 46:283-285 (2000); D. Borowitz etal., J. Pediatr., 127:681-684 (1995).

In the clinical setting, a number of manufacturers have begun to usestructured fats or structured lipids as a dietary source of fats.Structured fats or lipids are created by separating fatty acids from theglycerol backbone of medium- and long-chain triglycerides, a processcalled de-esterification. The generated fatty acids are then rejoinedthrough re-esterification to create triglycerides containing medium- andlong-chain fatty acids on the same glycerol backbone. Structured fats orlipids are limited in their effectiveness as nutrient supplement becausethe fats or lipids still need to be hydrolyzed by lipases so that thefatty acids and monoglycerides can be absorbed properly by the body.This random re-esterification used to create structured fats or lipidsmay not produce fats that are easily absorbable by the body, since there-esterification may occur at the incorrect glycerol backbone,potentially leaving the long-chain poly-unsaturated fats at theincorrect glycerol site.

In clinical practice, the average daily dose of porcine-derivedpancreatic enzyme capsules may vary from 17 to 50 capsules per day,which may need to be individualized due to the inherent variability ofthe porcine-derived pancreatic enzyme, polymeric coating, and foodconsumption, and for some patients, taking other drugs may significantlyaffect the quality of life. As the risk of malnutrition from not takingpancreatic enzymes, even with the high doses, is much greater than thepotential risk related to phthalates, it is advised that patients withCF continue to take their pancreatic enzymes as prescribed.Unfortunately, as previously noted, high doses of porcine pancreaticenzyme supplements have been found to be associated with fibrosingcolonopathy in patients with CF.

To supplement a required caloric intake and absorption of LC-PUFAs,patients with EPI and/or people having inadequate absorption of LC-PUFAsmay consume liquid nutritional formula through enteral feeding togetherwith the oral intake of the porcine-derived pancreatic enzyme capsulesin PERT. However, a timing gap between the nutritional liquid and theadministration of the porcine-derived pancreatic enzyme capsules and/ora lack of synchronization in the small intestine between theavailability of the enzymes released from the capsules and the use ofenteral formula can exist, which may lead to inefficient enzymaticactivity and thus reduced fat hydrolysis and absorption. For at leastthe above limitations combined, PERT fails to solve the problems ofinadequate absorption, maldigestion, and malabsorption of fats, inparticular LC-PUFAs, and may limit caloric intake, create fatty acidimbalances and/or deficiencies, exacerbate GI symptoms, require highvolumes of nutritional liquid, and thus may significantly affect qualityof life.

Accordingly, there exists a need for a device and a method fordelivering readily absorbable fats (free fatty acids andmonoglycerides), such as LC-PUFAs, to a person in need of the nutrient.In addition, there exists a need for a device and a method capable ofefficiently hydrolyzing long-chain triglycerides to deliver absorbablefats in the form of monoglycerides and free fatty acids directly to thegastrointestinal tract. Embodiments of the present disclosure describedherein aim to overcome one or more of the limitations of the currentlyavailable treatment options and to improve the quality of life forpeople having impaired ability to adequately hydrolyze dietary fats, forexample, LC-PUFAs.

SUMMARY OF THE DISCLOSURE

Embodiments of the present disclosure are directed to devices andmethods for hydrolyzing fats in a nutritional formula by exposing thenutritional formula to lipase directly before ingestion. Variousembodiments of the disclosure may include one or more of the followingaspects.

In accordance with one embodiment, an enteral feeding device forhydrolyzing triglycerides and fatty acid esters in a nutritional formulaby exposing the nutritional formula to lipase may include a body housinga chamber. The device may also include an inlet configured to fluidlycouple with a source tube, creating a pathway for the nutritionalformula to enter the device from the source tube and flow into thechamber. The device may also include an outlet configured to fluidlycouple with an enteral feeding tube, creating a pathway for thenutritional formula to exit the chamber and flow into the enteralfeeding tube. The device may also include a plurality of particlescontained within the chamber, wherein the lipase may be covalentlybonded to each of the plurality of particles. The device may alsoinclude an inlet filter located between the inlet and the chamber,wherein the inlet filter contains a first plurality of openingsconfigured to broaden a flow path of the nutritional formula as it flowsfrom the inlet and into the chamber. The device may also include anoutlet filter located between the chamber and the outlet, wherein theoutlet filter has a second plurality of openings, and wherein the secondplurality of openings are smaller than the plurality of particles. Thetriglycerides and fatty acid esters in the nutritional formula may behydrolyzed as they pass through the plurality of particles containedwithin the chamber.

Various embodiments of the enteral feeding device may include one ormore of the following features: the plurality of particles, when dry,may fill at least 50% of the chamber; the plurality of particles, whendry, may fill at least 80% of the chamber; the plurality of particles,when dry, may fill at least 90% of the chamber; the plurality ofparticles, when exposed to the nutritional formula, may fill at least80% of the chamber; the plurality of particles, when exposed to thenutritional formula, may fill at least 90% of the chamber; the pluralityof particles, when dry, may fill substantially the same amount of thechamber as when exposed to the nutritional formula; the plurality ofparticles may swell so that, when dry, the plurality of particles mayfill less of the chamber than when exposed to the nutritional formula;an outside surface of at least one of the plurality of particles may beat least partially hydrophobic; the device may be configured so thatthere is less than a 30% difference between a flow rate set by the pumpand a flow rate of the nutritional formula exiting the outlet; at leastone of the plurality of particles may be formed of one or more ofethylene glycol dimethacrylate, butyl methacrylate, or glycidylmethacrylate; at least one of the plurality of particles may be formedof between about 50% to about 60% of ethylene glycol dimethacrylate byweight; at least one of the plurality of particles may be formed ofbetween about 30% to about 45% of butyl methacrylate by weight; at leastone of the plurality of particles may be formed of between about 0.01%to about 10% of glycidyl methacrylate by weight; at least one of theplurality of particles may have a hydrophilic coating includingpolyethylene glycol; at least one of the plurality of particles may beformed of between about 0% to about 10% of polyethylene glycol byweight; at least one of the plurality of particles may have asubstantially solid cross-section; at least one of the plurality ofparticles may have a substantially smooth outer surface; at least one ofthe plurality of particles may have an irregular outer surface; at leastone of the plurality of particles may have a porous cross-sectionforming internal surfaces within the at least one particle; a median ora mean diameter of a pore of the porous cross-section may range fromabout 1 nm to about 50 nm; a median or a mean diameter of a pore of theporous cross-section may range from about 1 nm to about 50 μm; thelipase may be covalently bonded to the internal surfaces; at least oneof an outer surface or an internal surface of at least one of theplurality of particles may include a functional group; the functionalgroup may be an epoxy group; the lipase may be covalently bonded to theepoxy group; the lipase may be selected from at least one ofChromobacterium viscosum lipase, Pseudomonas fluorescens lipase, orRhizopus oryzae lipase; a median or a mean diameter of the plurality ofparticles may be between about 100 μm and about 800 μm; a median or amean diameter of the plurality of particles may be between about 200 μmand about 500 μm; the plurality of particles may include a first groupof particles and a second group of particles, wherein the first group ofparticles has a median or a mean diameter of that is different than amedian or a mean diameter of the second group of particles; an amount ofthe lipase covalently bonded to the plurality of particles may fallwithin a range of about 5 mg to about 500 mg of lipase per 1 g of theplurality of particles; an average size of at least one of the firstplurality of openings or the second plurality of openings may be betweenabout 10% to about 60% smaller than an average diameter of the pluralityof particles; at least one of the first plurality of openings or thesecond plurality of openings may include a plurality of tortuous paths;the inlet filter may be coated with at least one emulsifier configuredto emulsify the nutritional formula as it passes through the inletfilter; the inlet filter and the outlet filter each may have a thicknessof between about 0.1 mm to about 10 mm; and the device may be furtherconfigured to hydrolyze phospholipids.

It is to be understood that the present disclosure is not limited in itsapplication to the details of construction and to the arrangements ofthe components set forth in the following description or illustrated inthe drawings. The present disclosure is capable of embodiments inaddition to those described and of being practiced and carried out invarious ways. Also, it is to be understood that the phraseology andterminology employed herein, as well as the abstract, are for thepurpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be used as a basis fordesigning other structures, methods, and systems for carrying out theseveral purposes of the present disclosure. It is important, therefore,to recognize that the claims should be regarded as including suchequivalent constructions insofar as they do not depart from the spiritand scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate exemplary embodiments of thepresent disclosure, and together with the description, serve to explainthe principles of the disclosure.

FIG. 1 illustrates an exemplary system for supplying and processing anutritional formula, according to embodiments of the present disclosure.

FIG. 2 illustrates a cross-section of an exemplary fat hydrolysisdevice, according to embodiments of the present disclosure.

FIG. 3A illustrates a cross-section of an exemplary fat hydrolysisdevice, according to embodiments of the present disclosure.

FIG. 3B illustrates a perspective view of an exemplary fat hydrolysisdevice, according to embodiments of the present disclosure.

FIG. 4A illustrates a cross-section of an outlet of an exemplary fathydrolysis device, according to embodiments of the present disclosure.

FIG. 4B illustrates a magnified view of a portion of the outlet depictedin FIG. 4A.

FIG. 4C illustrates a perspective view of the outlet of FIG. 4A.

FIG. 5 is a scanning electron microscope image of exemplary particles,according to embodiments of the present disclosure.

FIG. 6A illustrates a cross-section of an exemplary fat hydrolysisdevice, according to embodiments of the present disclosure.

FIG. 6B illustrates a cross-section of an exemplary fat hydrolysisdevice, according to embodiments of the present disclosure.

FIG. 7A illustrates a magnified view of a surface of an exemplaryparticle, according to embodiments of the present disclosure.

FIG. 7B illustrates a magnified view of a surface of an exemplaryparticle, according to embodiments of the present disclosure.

FIG. 7C illustrates a magnified cross-section of an exemplary particle,according to embodiments of the present disclosure.

FIG. 7D illustrates a magnified cross-section of an exemplary particle,according to embodiments of the present disclosure.

FIG. 7E illustrates a magnified cross-section of an exemplary particle,according to embodiments of the present disclosure.

FIG. 7F illustrates a magnified cross-section of an exemplary particle,according to embodiments of the present disclosure.

FIG. 8A is a scanning electron microscope image of exemplary particles,according to embodiments of the present disclosure.

FIG. 8B is a scanning electron microscope image of a cross-section of anexemplary particle, according to embodiments of the present disclosure.

FIG. 9 is a scanning electron microscope image showing inner structuresof an exemplary particle, according to embodiments of the presentdisclosure.

FIG. 10A is a schematic representation of the crystal structure of anexemplary lipase molecule, according to embodiments of the presentdisclosure.

FIG. 10B is a schematic representation of an exemplary particle,according to embodiments of the present disclosure.

FIG. 10C is a schematic representation of a plurality of lipasemolecules from FIG. 10A bound the exemplary particle of FIG. 10B,according to embodiments of the present disclosure.

FIG. 10D illustrates a cross-section of an exemplary fat hydrolysisdevice containing a plurality of the bound particles of FIG. 10C,according to embodiments of the present disclosure.

FIG. 11 graphically compares specific activities of lipase attached toexemplary particles, according to embodiments of the present disclosure.

FIG. 12 graphically compares release of lipase from exemplary particles,according to embodiments of the present disclosure.

FIG. 13 graphically depicts the amount of free fatty acid generated in asample of enteral formula Peptamen AF® hydrolyzed by an exemplary fathydrolysis device, according to embodiments of the present disclosure.

FIG. 14 is a schematic representation of the hydrolysis of atriglyceride molecule by an exemplary lipase molecule, according toembodiments of the present disclosure.

FIG. 15 illustrates a magnified schematic view of an exemplary particle,according to embodiments of the present disclosure.

FIG. 16A illustrates a magnified schematic of a cross-section of anexemplary filter mesh material, according to embodiments of the presentdisclosure.

FIG. 16B illustrates a magnified schematic of a cross-section of anexemplary filter mesh material, according to embodiments of the presentdisclosure.

FIG. 17 illustrates the flow of nutritional formula through an exemplaryfat hydrolysis device at different time periods, according toembodiments of the present disclosure.

FIG. 18 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 19 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 20 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 21 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 22 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 23 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 24 graphically compares the flow rates of an exemplary nutritionalformula through exemplary enteral feeding circuits, according toembodiments of the present disclosure.

FIG. 25 graphically depicts the flow rates of an exemplary nutritionalformula through an exemplary fat hydrolysis device in three test runs,according to embodiments of the present disclosure.

FIG. 26 graphically depicts the flow rate of an exemplary nutritionalformula through an exemplary fat hydrolysis device, according toembodiments of the present disclosure.

FIG. 27 graphically depicts the flow rate of an exemplary nutritionalformula through an exemplary fat hydrolysis device over a 4-hoursimulated feeding period.

FIG. 28 graphically depicts the fat content and types of fat ofcommercially available enteral formulas, according to embodiments of thepresent disclosure.

FIG. 29 graphically depicts the percentage of fat hydrolyzed out of theexemplary enteral formulas of FIG. 16 using an exemplary fat hydrolysisdevice, according to embodiments of the present disclosure.

FIG. 30 graphically depicts accumulation of the amount of free fattyacid in a sample of enteral formula Peptamen AF® hydrolyzed by anexemplary fat hydrolysis device, according to embodiments of the presentdisclosure.

FIG. 31 graphically depicts accumulation of the amount of free fattyacid in a sample of enteral formula Peptamen AF® hydrolyzed by anexemplary fat hydrolysis device, according to embodiments of the presentdisclosure.

FIG. 32 graphically compares accumulation of the amount of free fattyacid in an exemplary nutritional formula achieved when usingcommercially available lipase supplements versus an exemplary fathydrolysis device, according to embodiments of the present disclosure.

FIG. 33 graphically compares calculated hydrolysis efficiencies of fatsin the three samples shown in FIG. 32.

FIG. 34 graphically depicts hydrolysis of fats from a representativecomplex nutritional formula during simulated feedings using an exemplaryfat hydrolysis device, according to embodiments of the presentdisclosure.

FIG. 35 schematically depicts the study design and procedures for the6-week pig study described in Example 13.

FIG. 36A shows stool appearance of pigs having exocrine pancreaticinsufficiency (EPI pigs) fed with non-hydrolyzed formula (“EPI”).

FIG. 36B shows stool appearance of EPI pigs fed with formulapre-hydrolyzed by an exemplary Rhizopus oryzae lipase attached toparticles (“EPI+iRO”).

FIG. 37 graphically compares fat measured in stool samples of healthy(“Healthy”), EPI, and EPI+iRO pigs.

FIG. 38A graphically compares mean of formula intake of Healthy, EPI,and EPI+iRO pigs.

FIG. 38B graphically compares mean of body weight of Healthy, EPI, andEPI+iRO pigs.

FIG. 39 graphically compares plasma polyunsaturated free fatty acidlevels of Healthy, EPI, and EPI+iRO pigs, measured in pre-prandial bloodsamples.

FIG. 40A graphically compares plasma polyunsaturated free fatty acidconcentration (mean±SD) in Healthy, EPI, and EPI+iRO pigs.

FIG. 40B graphically compares polyunsaturated free fatty acidconcentration (mean±SD) in Healthy, EPI, and EPI+iRO pigs, measured inpost-prandial samples.

FIG. 41 graphically compares mean accretion of AA and DHA in the heart,liver, fat, and hippocampus of Healthy, EPI, and EPI+iRO pigs.

FIG. 42 schematically depicts the study design and procedures for the12-day pig study described in Example 14.

FIG. 43 graphically compares the mean mucosal thickness of the smallintestine of the control group and the test group described in Example14.

FIG. 44 graphically compares the mean changes in DHA and EPA fastingplasma levels of the control group and the test group described inExample 12.

FIG. 45 graphically compares the lipid absorption measured from bloodsamples before and after solid meals of the control group and before andafter G-tube feeding of the test group described in Example 14.

FIG. 46 graphically compares mean coefficient of protein absorption ofthe control group and the test group described in Example 14.

FIG. 47 graphically compares mean fat absorption of EPI pigs of acontrol group fed non-hydrolyzed nutritional formula and a test groupfed nutritional formula pre-hydrolyzed with an exemplary fat hydrolysisdevice described in Example 14.

FIG. 48A graphically compares phramacodynamic profiles of EPA of thecontrol group and the test group described in Example 15.

FIG. 48B graphically compares phramacodynamic profiles of DHA of thecontrol group and the test group described in Example 15.

FIG. 49A graphically compares plasma levels over time of DHA and EPA ofthe control group and the test group described in Example 16.

FIG. 49B graphically compares the absolute increase in total DHA and EPAof the control group and the test group described in Example 16.

DETAILED DESCRIPTION

Reference will now be made in detail to the exemplary embodiments of thepresent disclosure described below and illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to same or like parts.

While the present disclosure is described herein with reference toillustrative embodiments of particular applications, such as devices,methods, and systems for supplying and processing nutritional formulasprior to ingestion, it is understood that the embodiments describedherein are not limited thereto. Those having ordinary skill in the artand access to the teachings provided herein will recognize additionalmodifications, applications, embodiments, and substitution ofequivalents that all fall within the scope of the present disclosure.For example, the devices and methods of the present disclosure may beemployed for any suitable application, including, but not limited to,supplying fatty acid needs for medical and nutritional purposes forinfants, children, or adults, in the hospital, in supportive careinstitutions, in long-term care facilities, or for home use, or forveterinary use, or for use with livestock. Devices disclosed herein canalso be used with other suitable fat-containing liquids. Accordingly,the disclosure is not to be considered as limited by the foregoing orfollowing descriptions.

FIG. 1 illustrates an exemplary embodiment of an enteral supply system100 for feeding a nutritional formula 110 to a subject via a feedingtube. System 100 may include a fat hydrolysis device 200, a pump 120,and a first tube 122 fluidly connecting a source of nutritional formula110 and device 200. Nutritional formula 110 may be contained in asuitable container, such as a feeding bag, a vial, a syringe, or abottle. Nutritional formula 110 is flowed from the source, through firsttube 122, and to device 200 for processing. System 100 also includes asecond tube 124 having an end configured to connect to device 200 and anopposite end configured to connect to a patient to deliver processednutritional formula 110 from device 200 to the patient for ingestion.Second tube 124 may be an enteral feeding tube, for example, a gastric,a nasogastric, a nasoduodenal, a nasojejunal, a gastrostomy, agastrojejunostomy, a jejunostomy, a PEG tube, or a transjejunal feedingtube to feed nutritional formula 110 to the GI tract of a subjectthrough, for example, the nose, mouth, stomach, or abdomen. System 100may be used in line with current standard enteral feeding practice.

System 100 is configured to deliver and process nutritional formula 110at the point of care to allow device 200 to hydrolyze fats contained innutritional formula 110 right before ingestion. As used herein, the term“nutritional formula” refers to complex mixtures containing, forexample, proteins, carbohydrates, fat, water, minerals, and/or vitamins,which may include liquid foods that are specially formulated andprocessed; liquids used for the partial or exclusive feeding of a personby means of oral intake or feeding by tube; liquids used for the dietarymanagement of a person who, because of therapeutic or medical need, haslimited or impaired capacity to ingest, digest, absorb, or metabolizeordinary foodstuffs or certain nutrients; liquids that meet medicallydetermined nutrient requirements; and liquids designed to deliver to asubject nutrients that cannot be provided to the subject via dietarymanagement and modification of the normal diet alone. Nutritionalformula 110 may also include formulas intended for the specific dietarymanagement of a disease or condition, for which distinctive nutritionalrequirements, based on recognized scientific principles, are establishedby medical evaluation, or may include liquid foods used as part of anoverall diet to manage the symptoms or reduce the risk of a disease orcondition. In some embodiments, nutritional formula 110 may be deliveredto the subject under medical supervision, may be intended only for aperson receiving active and ongoing medical supervision, or may bedelivered to the subject for home use, either when supervised orunsupervised.

Nutritional formula 110 may be packaged as a dry powder or oil and thenmixed with a solvent to form a solution. In other embodiments,nutritional formula 110 may be packaged as a liquid nutritional formula,beverage, or drink. In some embodiments, nutritional formula 110 may becommercially available, or may be prepared by a healthcare professionalbefore feeding. Nutritional formula 110 may be an infant and/or toddlerformula as a complete or partial substitute for human milk, may be donormilk or breast milk, or may be designed to supplement or completelyreplace the diet of an adult or elderly person. In some embodiments,nutritional formula 110 may be a commercially available or acustom-developed formula combined with a commercially available or acustom-developed supplement or fortifier, which may supply additionalnutrients including, but not limited to, one or more of LC-PUFAs,vitamin, minerals, or proteins. In some embodiments, nutritional formula110 may include a combination of MCTs and LCTs. In some embodiments,nutritional formula 110 may be conditioned to make fats contained in itmore accessible for hydrolysis. Exemplary conditioning may include oneor more of sonication, fat droplet disruption, or emulsification, e.g.,by physical or chemical means (e.g. by exposure to a surfactant,surfactant-like substance, or protease). In some embodiments,nutritional formula 110 may be prescribed for a subject in need ofadditional LC-PUFAs, such as DHA, EPA, and/or AA, a subject havingconditions such as maldigestion and malabsorption of lipids, reducedcaloric intake, significant weight loss, LC-PUFA deficiencies, and/or asubject having diseases, including cystic fibrosis (CF), chronicpancreatitis (CP), surgery, cancer, liver abnormalities,gastrointestinal dysfunction, and developmental immaturity. In someembodiments, the subject may have exocrine pancreatic insufficiency(EPI) with reduced ability to hydrolyze long-chain triglycerides. Insome embodiments, nutritional formula 110 may include at least onemedicament prescribed for the subject in need of the medicament and/ornutritional formula 110, or nutritional formula 110 may itself be theprescribed medicament.

Nutritional formula 110 includes at least one fat in triglyceride form,such as MCT and LCT. In some embodiments, nutritional formula 110 mayfurther include at least one nutrient selected from water, maltodextrin,protein, hydrolyzed protein, amino acids, peptides, medium chaintriglycerides, diglycerides, monoglycerides, cornstarch, fish oil,soybean oil, rapeseed oil, cottonseed oil, sunflower oil, olive oil(oils may or may not be refined), soluble fiber, lecithin, magnesiumchloride, sodium ascorbate, guar gum, calcium phosphate, salt, cholinechloride, phosphoric acid, calcium citrate, sodium phosphate, taurine,magnesium oxide, zinc sulfate, potassium chloride, niacinamide, ferroussulfate, calcium pantothenate, manganese sulfate, pyridoxinehydrochloride, copper sulfate, thiamine mononitrate, beta-carotene,riboflavin, vitamin a palmitate, folic acid, biotin, sodium selenate,chromium chloride, potassium iodide, sodium molybdate, soluble fiber,fructooligosaccharide, probiotic, citric acid, vitamin A, vitamin D,vitamin E, vitamin B₁, vitamin B₂, vitamin B₃, vitamin B₅, vitamin B₆,vitamin B₇, vitamin B₉, and vitamin B₁₂. Exemplary nutritional formulasand systems are described in International Patent Application No.PCT/US2013/026063, filed Feb. 14, 2013, and U.S. patent application Ser.No. 14/378,856, filed Aug. 14, 2014, both of which are hereinincorporated by reference in their entireties.

The flow of nutritional formula 110 to device 200, and ultimately to thesubject, is controlled by pump 120 of system 100. In some embodiments,pump 120 may be a peristaltic pump, although any suitable type ofinfusion pump, e.g., an elastomeric pump, a multi-channel pump, asyringe pump, and/or a smart pump may be used. A flow rate ofnutritional formula 110 through the tubes and/or device 200 may be setand/or adjusted by pump 120. In some embodiments, pump 120 may include aprocessor, a display, and/or actuators (e.g. buttons, knobs, touchscreen, etc.) to adjust and control the flow rate of nutritional formula110 in system 100 and device 200. Pump 120 may be adjusted and set by ahealthcare provider and/or the subject receiving nutritional formula110. Pump 120 may perform continuous feeding, pulsatile feeding,intermittent feeding, bolus feeding, and/or flushing, and delivery offluids may be set or adjusted automatically, semi-automatically, ormanually.

In some embodiments, pump 120 may be a smart pump. Pump 120 may makeautomatic adjustments to the flow rate based on timing or feedback fromsystem 100. Pump 120 may include user alerts to warn when the user setsparameters for pump 120 that fall outside of specified limits. Pump 120may send an alert when an actual flow rate of nutritional formula 110falls outside of set parameters for pump 120. The parameters may bestored in a memory of pump 120, or may be entered and/or adjusted for aspecific delivery regime.

In other embodiments, system 100 may not include pump 120 and mayinstead depend on gravity to flow nutritional formula 110 through device200. The relative positioning of the source of nutritional formula 110may allow nutritional formula 110 to flow through the tubes and device200 under the influence of gravity alone. For example, a container ofnutritional formula 110 may be placed above device 200 and/or above thesubject, as shown in FIG. 1.

In other embodiments, pump 120 may be replaced with a syringe. Thesyringe may be filled with nutritional formula 110, and the flow rate ofnutritional formula 110 in the tubes or device 200 may be set, and/oradjusted by using the syringe manually, semi-automatically, orautomatically. For example, nutritional formula 110 may be pre-packagedin a pre-filled syringe mounted inside of an auto-injector-like device.The pre-packaged formula may also contain a pump ‘engine’ (e.g., aspring-loaded piston), and may be used to deliver the formula throughdevice 200 and to the feed tube.

In other embodiments, system 100 may use any suitable means, e.g., aballoon or other suitable pressure-generating device, to generate apressure drop or a flow-driving force that drives nutritional formula110 through the tubes and/or device 200.

FIG. 2 illustrates an exemplary device 200 in accordance with thepresent disclosure. Device 200 may include a body 210 having an inlet212, a chamber 222, and an outlet 230. Chamber 222 may contain aplurality of particles 300. Device 200 may further include a firstconnector 240 and a second connector 270 configured to fluidly connectwith first tube 122 and enteral tube 124, respectively. In someembodiments, device 200 may include an inlet filter 250 and an outletfilter 260. For example, inlet filter 250 may be located adjacent inlet212, and outlet filter 260 may be located adjacent outlet 230. In someembodiments, inlet filter 250 and outlet filter 260 may cooperativelydefine chamber 222 while in some embodiments, either or both of inletfilter 250 and outlet filter 260 may be located within or outside ofchamber 222. For example, there may be a floor and a ceiling thatcooperatively define chamber 222. The floor and ceiling may define oneor more openings at the top and bottom of chamber 222 and/or they may beporous to allow fluid to pass through into chamber 222. Inlet filter 250may be located above an opening in the ceiling of chamber 222 adjacentinlet 212 and/or outlet filter 260 may be located below an opening inthe floor of chamber 222 adjacent outlet 230. In some embodiments, inletfilter 250 may be located below a ceiling within chamber 222 and/oroutlet filter 260 may be located above a floor within chamber 222, orany combination of positions thereof. Inlet filter 250 and outlet filter260 may prevent particles 300 from exiting device 200. Additionally oralternatively, the filters may prevent foreign objects from enteringdevice 200 and/or enteral tube 124. Particles 300 may be located betweeninlet filter 250 and outlet filter 260 in chamber 222. Inlet filter 250and outlet filter 260 may retain particles 300 within chamber 222 asnutritional formula 110 flows through device 200. The smaller poreopenings in inlet filter 250 and/or outlet filter 260 may aid in theemulsification and breakdown of fats.

As shown in FIG. 3A, body 210 may include one or more additionalchambers. For example, body 210 may include an inlet chamber 214, aninlet filter chamber 218 for holding inlet filter 250, an outlet filterchamber 224 for holding outlet filter 260, and/or an outlet chamber 228.In some embodiments, the perimeter of inlet filter 250 may be about thesame shape and size as that of the interior perimeter of inlet filterchamber 218. Inlet filter 250 may be fixed in inlet filter chamber 218via, e.g., friction fit, press fit, snap fit, twist fit, and/orultrasonic welding. In some embodiments, the perimeter of inlet filterchamber 218 may be smaller than the interior perimeter of chamber 222.In some embodiments, the perimeter of inlet filter chamber 218 may belarger than that of chamber 222 such that an edge portion may exist toallow inlet filter 250 be held against and/or out of chamber 222. Inother embodiments, inlet filter 250 may be placed in inlet chamber 214or inlet 212. In some embodiments, inlet chamber 214 may be shaped as anupside-down funnel, widening as it extends away from inlet 212. Theinterior perimeter of the wide end of inlet chamber 214 may be smallerthan the perimeter of inlet filter chamber 218 such that an edge 220 mayhold inlet filter 250 against inlet chamber 214. In other embodiments,device 200 may not include inlet chamber 214.

The placement of outlet filter 260 may have similar configurations asthat of inlet filter 250. For example, in some embodiments, theperimeter of outlet filter 260 may be about the same shape and size asthe interior perimeter of outlet filter chamber 224. Outlet filter 260may be fixed in outlet filter chamber 224 via, e.g., friction fit, pressfit, snap fit, twist fit, and/or ultrasonic welding. In someembodiments, the interior perimeter of outlet filter chamber 224 may belarger than that of chamber 222 such that an edge 226 may hold outletfilter 260 against and/or out of chamber 222. In other embodiments,outlet filter 260 may be located in outlet chamber 228. In someembodiments, the interior perimeter of outlet chamber 228 may be smallerthan the interior perimeter of outlet filter chamber 224 such that anedge portion may hold outlet filter 260 against and/or out of outletchamber 228. In other embodiments, body 210 may not include outletchamber 228.

In one embodiment, the interior region of body 210 may be shaped as ahollow cylinder. In another embodiment, the interior region of body 210may be shaped as, for example, a hollow truncated cone or a hollowpolygonal prism (such as a triangular, rectangular, pentagonal,hexagonal, or decagonal prism). The perimeter may be consistent in sizealong the length of device 200 or may vary, e.g., taper and/or flare.The walls may be smooth or textured. Different interior portions ofdevice 200 may have different shapes or texturing. In FIG. 3B, theexterior surface of body 210 is shaped as a polygonal prism, althoughthe exterior may have any suitable shape, e.g., cylindrical, polygonal,etc. The exterior surface may have one or more textured areas, surfaces,indentations, or ridges to provide easy handling or gripping by a user.As noted in FIGS. 3A and 3B, the interior and exterior shape may not bethe same, although in other embodiments, they may. Body 210 may be anysuitable shape and include at least one chamber 222. In exemplaryembodiments, chamber 222 may have a circular or ellipticalcross-section. In exemplary embodiments, more than one chamber 222 maybe included in body 210, arranged in series or in parallel, and may befluidly connected.

In some embodiments, the interior diameter of a cross-section of body210 may range from about 0.5 cm to about 1.5 cm, from about 0.5 cm toabout 2 cm, from about 1.5 cm to about 1.7 cm, from about 2 cm to about4 cm, from about 4 cm to about 6 cm, from about 6 cm to about 8 cm, fromabout 8 cm to about 12 cm, or from about 12 cm to about 15 cm. In someembodiments, the diameter of a cross-section of body 210 may decrease orincrease along the length of body 210 by a range from about 1% to about5%, from about 5% to about 10% from, from about 10% to about 20%, fromabout 20% to about 30%, from about 30% to about 40%, from about 40% toabout 50%, from about 1% to about 10%, from about 1% to about 20%, fromabout 1% to about 30%, from about 1% to about 40%, or about 1% to about50%. The length of body 210 may range from about 1 cm to about 5 cm,from about 2 cm to about 6 cm, from about 4 cm to about 6 cm, from about4 cm to about 8 cm, from about 1 cm to about 6 cm, from about 1 cm toabout 8 cm, or from about 1 cm to about 10 cm, and the total length ofdevice 200 may range from about 1.5 cm to about 6.5 cm, from about 2 cmto about 6.5 cm, from about 4.5 cm to about 6.5 cm, from about 4.5 cm toabout 8.5 cm, from about 1.5 cm to about 6.5 cm, from about 1.5 cm toabout 8.5 cm, from about 1.5 cm to about 12.5 cm, from about 2.5 cm toabout 15 cm, from about 4.5 cm to about 15 cm, from about 6.5 cm toabout 15 cm, from about 8.5 cm to about 15 cm, from about 10 cm to about15 cm, or from about 1.5 cm to about 15 cm. In some embodiments, thevolume of chamber 222 may range from about 0.5 mL to about 2 mL, fromabout 2 mL to about 5 mL, from about 4 mL to about 6 mL, from about 5 mLto about 8 mL, from about 5 mL to about 10 mL, from about 10 mL to about15 mL, from about 15 mL to about 20 mL, from about 25 mL to about 30 mL,from about 0.5 mL to about 4 mL, from about 0.5 mL to about 5 mL, fromabout 0.5 mL to about 6 mL, from about 0.5 mL to about 8 mL, from about0.5 mL to about 10 mL, from about 0.5 mL to about 15 mL, from about 0.5mL to about 20 mL, from about 0.5 mL to about 25 mL, or from about 0.5mL to about 30 mL.

In some embodiments, inlet filter 250 and outlet filter 260 may form atop end and a bottom end of chamber 222, respectively. In suchembodiments, the location of chamber 222 along a longitudinal axis ofbody 210 and/or the volume of chamber 222 may be adjusted by adjustingthe location of inlet filter 250 and/or outlet filter 260 within body210. In some embodiments, the total volume inside body 210 may rangefrom about 0.5 mL to about 2 mL, from about 2 mL to about 5 mL, fromabout 5 mL to about 10 mL, from about 10 mL to about 15 mL, from about15 mL to about 20 mL, from about 25 mL to about 30 mL, from about 0.5 mLto about 10 mL, from about 0.5 mL to about 15 mL, from about 0.5 mL toabout 20 mL, from about 0.5 mL to about 25 mL, or from about 0.5 mL toabout 30 m L.

In some embodiments, device 200 may include a first connector 240configured to connect first tube 122 to body 210 to deliver nutritionalformula 110 to device 200. First connector 240 may include an inlet 242to receive nutritional formula 110, an outlet 246, and a channel 244fluidly connecting the two. First connector 240 may include a fittingportion 248 configured to attach first connector 240 to body 210. Insome embodiments, inlet 242 may generally be in the shape of a cylinder,a funnel, or a truncated cone, and may be designed to match any suitablestandardized connector, such as an ENFit™ connector. In someembodiments, channel 244 may fluidly connect inlet 242 to inlet 212 ofbody 210. In some embodiments, inlet 242, or inlet 242 and channel 244,may form a female fitting configured to fit with a male fittingconnected to first tube 122. Or, an outer surface of first connector 240may form a male fitting configured to fit with a female fittingconnected to first tube 122. The male and female fittings may fit viaany suitable mechanical means, e.g., friction fit, press fit, twist fit,snap fit, overmolding or molding, thermal bonding, adhesive bonding,and/or welding. Indeed, first connector 240 may have any suitable sizeand shape for connecting device 200 to tube 122.

In some embodiments, body 210 may comprise a recessed portion 216, andfitting portion 248 of first connector 240 may form a complimentaryprotrusion, or vice versa, to connect the two parts. First connector 240may connect to body 210 via friction fit, twist fit, snap fit, clasp,press fit, overmolding or molding, thermal bonding, adhesive bonding,and/or welding. For example, first connector 240 may be pushed and/ortwisted against body 210 until fitting portion 248 abuts recessedportion 216. In other embodiments, first connector 240 and body 210 mayconnect via a screw mechanism. For example, first connector 240 and body210 may comprise a set of complementary screw threads such that firstconnector 240 may be fastened to body 210 by screwing first connector240 into body 210. In some embodiments, the perimeter of an outside wallof inlet 212 may be larger than that of an interior perimeter of channel244. For example, as shown in FIG. 3A, a rim of inlet 212 may be pushedor may abut an opening of channel 244 when first connector 240 and body210 are properly fitted and connected. Once inlet 242 and channel 244 offirst connector 240 and inlet 212 of body 210 are fluidly connected,nutritional formula 110 may flow from first tube 122, through firstconnector 240, and into body 210.

Although FIG. 3A depicts a separate first connector 240, in someembodiments, body 210 may connect directly to first tube 122, and aseparate first connector 240 may not be needed. Although the fluid pathis shown as extending through inlet 242, channel 244, and inlet 212, itis contemplated that this path may also include other portions or may beformed as a single portion in other embodiments.

In some embodiments, the diameter of inlet 242 may range from about 4 mmto about 7 mm, from about 5 mm to about 10 mm, or from about 4 mm toabout 10 mm; the diameter of inlet 212 may range from about 1 mm toabout 3 mm, from about 2 mm to about 4 mm, from about 3 mm to about 5mm, or from about 1 mm to about 5 mm; the diameter of inlet filterchamber 214 may range from about 8 mm to about 12 mm, from about 12 mmto about 15 mm, from about 15 mm to about 18 mm, or from about 8 mm toabout 18 mm; the diameter of outlet filter chamber 224 may range fromabout 10 mm to about 14 mm, from about 14 mm to about 17 mm, from about17 mm to about 20 mm, or from about 10 mm to about 20 mm; the diameterof outlet 230 may range from about 10 mm to about 15 mm, from about 15mm to about 20 mm, from about 20 mm to about 25 mm, or from about 10 mmto about 25 mm; and the diameter of fitting channel 234 may range fromabout 12 mm to about 16 mm, from about 16 mm to about 20 mm, from about20 mm to about 24 mm, from about 24 mm to about 28 mm, or from about 12mm to about 28 mm.

In some embodiments, a second connector 270 may be used to connectdevice 200 to enteral tube 124. Body 210 may comprise a rim 232encircling outlet 230, and rim 232 may have a fitting channel 234 forconnecting body 210 to second connector 270. As shown in FIG. 4A, secondconnector 270 may comprise an inlet 272, an inlet chamber 274, and anoutlet 282. In some embodiments, second connector 270 may comprise abrim 276 and a protrusion element 278 projecting up from brim 276towards inlet 272. In some embodiments, second connector 270 may connectto body 210 via friction fit, press fit, twist fit, clasp, snap fit,overmolding/molding, thermal bonding, adhesive bonding, and/or welding.For example, an outer perimeter of inlet chamber 274 of second connector270 may correspond in size and shape to an inner perimeter of outletchamber 228 of body 210 such that inlet chamber 274 of second connector270 may be pushed, twisted, or otherwise received within outlet chamber228 of body 210. In some embodiments, brim 276 may be pushed and/or mayabut rim 232 of body 210, and protrusion element 278 may fit intofitting channel 234. As shown in FIG. 4B, a cross-section of protrusionelement 278 is tapered and complements the tapered shape of fittingchannel 234, however, protrusion element 278 may have any suitableshape, for example, rectangular, triangular, semi-circular, polygonal,flared, bulbous, or conical, with predetermined dimensions and anglesfor mating with fitting channel 234. In some embodiments, the perimeterof the cross-section of fitting channel 234 and protrusion element 278may be similarly shaped or complementary.

In some embodiments, the perimeter of inlet 272 of second connector 270and outlet 230 of body 210 may be similarly shaped, for example,circular, elliptical, rectangular, pentagonal, or hexagonal, and maymate with each other. In some embodiments, as shown in FIG. 4C, secondconnector 270 may be a male connector having one or more stepped tubularportions. The stepped tubular portions may be shaped as hollow cylindersor hollow truncated cones, whose exterior perimeters decrease with eachadditional step. Second connector 270 may be configured to connect to afemale fitting of enteral tube 124. For example, the stepped tubularportions of second connector 270 may fit into a recess of a femaleconnector of enteral tube 124 via, e.g., friction fit, twist fit, snapfit, clasp, and/or press fit. In other embodiments, second connector 270may have any suitable shape, e.g., a cone, a truncated cone, or acylinder, and may be designed to match any suitable standardizedconnector, such as an ENFit™ connector, and may be smooth or may includeone or more ridges to facilitate connection to enteral tube 124. In someembodiments, second connector 270 may be a female portion for connectingto a male portion of enteral tube 124. Indeed, second connector 270 mayhave any suitable size and shape for connecting device 200 to enteraltube 124.

In some embodiments, at least one of first connector 240 and secondconnector 270 may be any suitable standardized connector, such as anENFit™ connector.

In some embodiments, the diameter of fitting channel 234 may range fromabout 12 mm to about 16 mm, from about 16 mm to about 20 mm, from about20 mm to about 24 mm, from about 24 mm to about 28 mm, or from about 12mm to about 28 mm; the interior diameters of inlet 272 and inlet chamber274 may range from about 4.5 mm to about 8 mm, from about 8 mm to about13 mm, from about 13 mm to about 15 mm, from about 15 mm to about 18 mm,or from about 4.5 mm to about 18 mm, and the exterior diameters of inlet272 and inlet chamber 274 may range from about 6 mm to about 10 mm, fromabout 10 mm to about 14 mm, from about 14 mm to about 18 mm, from about18 mm to about 21 mm, or from about 6 mm to about 21 mm; the diameter ofoutlet 282 may range from about 0.5 mm to about 1.5, from about 1.5 mmto about 2.5 mm, from about 2.5 mm to about 3.5 mm, or from about 0.5 mmto about 3.5 mm; and the diameter of brim 276 may range from about 7 mmto about 10 mm, from about 10 mm to about 15 mm, from about 15 mm toabout 20 mm, from about 20 mm to about 25 mm, from about 22 mm to about26 mm, from about 25 mm to about 30 mm, or from about 7 mm to about 30mm.

Although FIGS. 4A-4C depict a separate second connector 270; secondconnector 270 may not be a separate element of body 210. For example,second connector 270 may be formed integrally as part of body 210 suchthat body 210 directly connects with enteral tube 124. Although thefluid path is shown as extending through outlet 230, inlet 272, inletchamber 274, and outlet 282, it is contemplated that this path may alsoinclude other portions or may be formed as a single portion in otherembodiments.

In some embodiments, the dimensions or sizes of device 200 may beselected based on the particular application of device 200. For example,the diameters of inlet 212, inlet 242, inlet filter 250, chamber 222,outlet filter 260, and outlet 282, and the lengths and sizes of chamber222 and body 210 may be selected for feeding a nutritional formula 110to a particular subject. For example, the dimensions or sizes of device200 for feeding infants may be smaller than those of a device forfeeding youths and adults. In some embodiments, the dimensions or sizesof device 200 may be selected based at least in part on the amount oftime the device is intended to be used to feed nutritional formula 110to a subject, a flow rate or a volume of nutritional formula 110 to befed to a subject, or whether the device is intended to be attached to apump or not. For example, the dimensions or sizes of device 200 for anovernight enteral feeding procedure may be smaller than those for ashorter or faster enteral feeding procedure of nutritional formula 110,or a larger device may be used for a larger volume or faster intendedflow rate of nutritional formula 110.

In some embodiments, more than one device 200 may be connected inseries. For example, second connector 270 of a first device 200 may beconnected to first connector 240 of a second device 200. For anotherexample, a first end of a tube may be connected to second connector 270of a first device 200 and connected to first connector 240 of a seconddevice 200, allowing nutritional formula to flow from the first device200 to the second device 200.

In some embodiments, body 210, first connector 240, and second connector270 of device 200 may be made of the same material. In some embodiments,body 210, first connector 240, and second connector 270 of device 200may be made of different materials having different physical,mechanical, or chemical characteristics, such as, for example,flexibility, elasticity, tensile strength, toughness, color,transparency, chemical resistance, and/or thermal resistance, or theparts may be formed of a combination of materials. In some embodiments,the material of device 200 may be a medical grade biocompatible plastic.In some embodiments, device 200 may be sterilizable, and the material ofdevice 200 may be an autoclavable plastic, for example, polyethylene,polypropylene, or polycarbonate. In some embodiments, body 210, firstconnector 240, and second connector 270, may be manufactured viainjection molding or additive manufacturing techniques, such as 3Dprinting.

In one exemplary embodiment, body 210 of device 200 is made of a clearplastic so that the plurality of particles 300 inside chamber 222 ofbody 210 are visible to the user. Particles 300 contained in device 200have lipase immobilized on their surfaces, and as nutritional formula110 flows through chamber 222 and particles 300, the immobilized lipasehydrolyzes the fats and triglycerides, including triglycerides havingLC-PUFAs, in nutritional formula 110, breaking them down intomonoglycerides and free fatty acids. Particles 300 contained in chamber222 are discussed in detail further below.

As shown in FIG. 5, in some embodiments, an exemplary particle 310 ofparticles 300 may be formed as a substantially spherical bead. In someembodiments, particle 310 may have a diameter ranging from about 100 μmto about 800 μm, from about 100 μm to about 700 μm, from about 100 μm toabout 600 μm, from about 100 μm to about 500 μm, from about 100 μm toabout 400 μm, from about 100 μm to about 300 μm, from about 100 μm toabout 200 μm, from about 200 μm to about 800 μm, from about 200 μm toabout 700 μm, from about 200 μm to about 600 μm, from about 200 μm toabout 500 μm, from about 200 μm to about 400 μm, from about 200 μm toabout 300 μm, from about 300 μm to about 800 μm, from about 300 μm toabout 700 μm, from about 300 μm to about 600 μm, from about 300 μm toabout 500 μm, from about 300 μm to about 400 μm, from about 400 μm toabout 800 μm, from about 400 μm to about 700 μm, from about 400 μm toabout 600 μm, from about 400 μm to about 500 μm, from about 500 μm toabout 800 μm, from about 500 μm to about 700 μm, from about 500 μm toabout 600 μm, or from about 600 μm to about 800 μm. In otherembodiments, particle 310 may be a randomly shaped or irregularparticle, or may be elliptical, oblong, donut-shaped, a prism,polygonal, elongated, or any other suitable shape. Particle 310 may havea smooth or a textured surface. Particle 310 may be shaped to increaseor decrease its surface area. Particles 300 may be formed of individualparticles 310, which may each have substantially the same shape and/orsurface or may have two or more different shape and/or surfacecombinations.

In some embodiments, particles 300 have about the same diameter.Alternatively, particles 300 may have different diameters following askewed or a normal distribution. In some embodiments, the averagediameter of particles 300 may range from about 250 μm to about 500μm—for example, approximately 260 μm or approximately 460 μm—and mayfollow a normal distribution. In some embodiments, a skewed distributionof the diameters of particles 300 may have a mean diameter or a mediandiameter falling between about 100 μm and about 800 μm. In someembodiments, particles 300 may be pre-selected by a sieving process tofilter out particles having diameters smaller than a lower sizethreshold and/or larger than an upper size threshold. Sieving may allowfor more control and manipulation of the size and size distribution ofparticles 300. For example, particles 300 selected from one sievingprocess may have a narrower distribution of diameters and/or a larger orsmaller mean or median diameter compared to those of particles 300before the sieving process. In some embodiments, a mean or mediandiameter of particles 300 may be about 100 μm, about 150 μm, about 200μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450μm, or about 500 μm. In some embodiments, fines (much smaller particles,e.g., having diameters of less than approximately 50 μm) may be present,while in others, the fines may be absent from particles 300 included indevice 200. Fines may be manufactured or may occur during themanufacture of larger particles 300, e.g., as a result of the recipe oras a result of hydrodynamics of a reactor vessel. Fines may occur whilemanufacturing larger particles 300 and may be removed or left in themixture of particles, or fines may be manufactured separately and addedto larger particles 300, e.g., to increase the total surface area perunit volume or to allow proper flow rate, which, in some embodiments,may increase hydrolysis efficiency.

In some embodiments, particles 300 may be formed of differentsub-portions of particles, and each sub-portion may have a differentmedian or mean diameter or a different distribution of diameters. Forexample, in such embodiments, particles 300 may have a bimodal ormulti-modal distribution.

Particles 300 may be made of any suitable material, e.g., a polymericmaterial, a metal, etc. In some embodiments, particles 300 may be madeof acrylate polymers or acrylics. In some embodiments, particles 300 maybe made of a copolymer formed of multiple different monomers. Forexample, particles 300 may be made of a copolymer having three monomers,such as ethylene glycol dimethacrylate (EGDMA), butyl methacrylate(BMA), and glycidyl methacrylate (GMA). In some embodiments, EGDMA mayrange from about 25% to about 99% by weight, for example, from about 50%to about 60% by weight, of the composition of the copolymer. In someembodiments, BMA may range from about 1% to about 75% by weight, forexample, from about 30% to about 45% by weight, of the composition ofthe copolymer. Exemplary embodiments may contain EGDMA and BMA levels of90% and 9%, respectively; 60% and 39%, respectively; or 58% and 41%,respectively. In some embodiments, GMA may range from about 0.01% toabout 0.1%, from about 0.1% to about 1%, from about 1% to about 2%, fromabout 2% to about 5%, from about 5% to about 8%, from about 8% to about10%, from about 10% to about 15%, from about 15% to about 20%, fromabout 0.01% to about 10%, from about 0.01% to about 15%, or from about0.01% to about 20% by weight of the composition of the copolymer.Exemplary embodiments may contain epoxide levels (e.g., GMA) of 0%,0.25%, 1%, 2%, or 5%.

In some embodiments, particles 300 may be made of styrene polymers orstyrenes, caprolactone polymers or caprolactone, polydivinylbenzenepolymers or polydivinylbenzene, polyamides polymers or polyamides,polycarbonate polymers or polycarbonates, polypropylene polymers orpolypropylene, polyurethane polymers or polyurethane, polyethylenepolymers or polyethylene, methacrylate polymers or methacrylates,divinylbenzene (DVB) polymers or divinylbenzene, or of silica.Additional exemplary types of polymers suitable for making particles 300may include one or more selected from polymethacrylate, polyacrylate,polyurethane, polycarbonate, polydivinylbenzene, caprolactone,polystyrene, polyethylene, polypropylene, polyurethane, polyamides, andpolydivinylbenzene monomers, for example.

In some embodiments, particles 300 may be pre-selected and packaged indevice 200 during manufacture of device 200. In some embodiments,particles 300 may be packaged under dry conditions and placed in chamber222 of device 200 before being used in system 100. For example, thesize, type, or size distribution of particles 300 may be altered orselected depending on the intended use of device 200, and a user maypackage the necessary particles 300 in device 200 depending on thatspecific use. A moisture level of particles 300 upon being manufacturedand/or packaged in device 200 may range from about 0.1% to about 1%,from about 1% to about 2%, from about 2% to about 3%, from about 3% toabout 4%, from about 4% to about 5%, from about 0.1% to about 2%, fromabout 0.1% to about 3%, from about 0.1% to about 4%, or from about 0.1%to about 5% of water in the total composition of particles 300.

In some embodiments, the polymeric material of particles 300 may beinsoluble in acidic, basic, aqueous, and/or organic solvents. In someembodiments, particles 300 may be dispersed or suspended in an aqueoussolvent, an organic solvent, and/or an emulsion, for example, such as anoil-in-water or water-in-oil emulsion. In exemplary embodiments, whennutritional formula 110 is driven by pump 120 or by gravity to flowthrough chamber 222, particles 300 may be dispersed or suspended innutritional formula 110, and may move under the influence of the flowdynamics of nutritional formula 110 and/or random Brownian motion.

In some embodiments, particles 300 may swell upon being dispersed orsuspended in a solvent. As described herein, swelling of particles 300may refer to an increase in volume of particles 300, at least in part,due to absorption of the solvent by particles 300. Depending on thecomposition of particles 300 (e.g., polymeric material), the porosity ofparticles 300, and/or the composition of the solvent, particles 300 mayswell to different degrees when wetted. For example, the amount ofswelling may vary depending on solution conditions. Bead swelling may begreater in polar solvents, such as ethanol or acetone, whereas beadswelling may be less in water and water-based solutions. For example,particles 300 may swell by about 1% to about 25% in aqueous solutionsand by about 50% to about 100% in organic solvents, such as, forexample, ethanol, isopropanol, or acetone. In some embodiments, whenparticles 300 are dispersed or suspended in nutritional formula 110, theamount of swelling of particles 300 may depend on the composition, suchas fat content, protein content, vitamin content, ion content, etc., ofnutritional formula 110. In some embodiments, the amount of swelling ofparticles 300 in nutritional formula 110 may be minimal or none. In someembodiments, the amount of swelling of particles 300 in nutritionalformula 110 may be less than about 1%, about 2%, about 5%, about 10%,about 20%, about 30%, about 40%, or about 50% of the original, dryvolume.

As shown in FIG. 6A, in some embodiments, chamber 222 of body 210 mayinclude a headspace 223 that is not occupied by particles 300 under dryconditions. Chamber 222 may be filled with particles 300 when particles300 are dry, e.g., contain less that 5% water by weight. For example,when particles 300 are dry before nutritional formula 110 is flowed intochamber 222, headspace 223 may take up from about 0 to about 5%, fromabout 5% to about 10%, from about 5% to about 15%, from about 10% toabout 15%, from about 15% to about 20%, from about 20% to about 30%,from about 30% to about 40%, from about 40% to about 50%, from about 5%to about 20%, from about 5% to about 30%, from about 5% to about 40%,from about 5% to about 50%, or from about 0 to about 50% of the volumeof chamber 222. The initial, dry volume of headspace 223 depends on thenumber or volume of particles 300 packaged in chamber 222 and, in someembodiments, may be selected based on the propensity for particles 300to swell when exposed to liquid.

Swelling may affect the number of particles 300 included in chamber 222and/or the fill level of chamber 222. In embodiments in which particles300 have a propensity to swell, adequate head space may be left inchamber 222 when dry particles 300 are loaded into chamber 222 to allowroom for swelling to occur once a nutritional formula is introduced intodevice 200 and particles 300 are wetted. Devices with insufficientheadspace above particles 300 in chamber 222 may have increased risk offlow obstruction as particles 300 swell, causing an increase in pressureagainst inlet and outlet filters 250, 260 that contain particles 300within chamber 222. Depending on the material used to form particles300, the propensity for swelling may be higher or lower depending on thetype of nutritional formula 110 used. In some embodiments, the volume ofheadspace 223 prior to use may depend on the composition of nutritionalformula 110. And, in some embodiments, the type of particles 300 orvolume of headspace 223 may at least in part be selected according tothe type of nutritional formula 110 that device 200 will be used with.

As shown in FIG. 6B, in some embodiments, when nutritional formula 110is flowed through chamber 222, headspace 223 may be occupied bynutritional formula 110 with particles 300 suspended therein. Forexample, when nutritional formula 110 flows through chamber 222,particles 300 may mix with nutritional formula 110 and may move innutritional formula 110 so that the volume of headspace 223 that was notoccupied by particles 300 under dry conditions may be filled asnutritional formula 110 and particles 300 disperse to fill chamber 222.Incorporating headspace 223 may give particles 300 space in chamber 222to be mobile and to move and/or mix with nutritional formula 110 underthe influence of the flow dynamics of nutritional formula 110. In someembodiments, including headspace 223 may facilitate a reduction inchanneling or shunting or facilitate the distribution of nutritionalformula 110 through the particles by allowing particles 300 to move,flow, and/or mix, rather than becoming packed against outlet filter 260.Alternatively, including too much headspace 223 may also lead tochanneling of nutritional formula 110 around particles 300, particularlywhen device 200 is oriented horizontally. For example, when device 200is positioned horizontally, particles 300 may float to the top ofnutritional formula 110, leaving a channel beneath particles 300. As aresult, nutritional formula 110 may channel and flow under particles300, potentially reducing hydrolysis efficiency. Additionally, byreducing the amount of particles 300 in device 200 to provide moreheadspace, the amount of lipase in device 200 is also reduced, since thelipase is bound to particles 300. As a result, too much headspace 223may cause decreased effective hydrolysis for a given amount ofnutritional formula 110, because it is the lipase bound to particles 300that breaks down nutritional formula 110. Leaving too much headspace 223means fewer particles 300 are contained within chamber 222, and thusless lipase is contained in chamber 222, leaving too few particles 300to hydrolyze all of, or a majority of, the triglycerides in nutritionalformula 110.

In some embodiments, particles 300 undergo minimal or no swelling whensuspended in nutritional formula 110, and thus when nutritional formula110 flows through chamber 222, the space for particles 300 to move inchamber 222 may be substantially the same as the volume of headspace 223initially in chamber 222. In some embodiments, particles 300 may swellwhen exposed to in nutritional formula 110, and thus when nutritionalformula 110 flows through chamber 222, the swelling of particles 300 maypartially reduce the space for particles 300 to move within chamber 222.For example, if under dry conditions, headspace 223 takes up about 10%of the volume of chamber 222 and particles 300 take up about 90% of thevolume of chamber 222, when nutritional formula 110 flows throughchamber 222, swelling of particles 300 may cause particles 300 to takeup an additional 5% of the volume of chamber 222, reducing the spaceleft for particles 300 to move to about 5% of the volume of chamber 222.Having more space for particles 300 to move may increase the mobility ofparticles 300 in chamber 222. Thus, in some embodiments, the swelling ofparticles 300 may reduce the mobility of particles 300 compared to theoriginal, dry volume.

In some embodiments, as shown in FIG. 6B, upon swelling, particles 300may become packed, resulting in friction between the surfaces ofparticles 300, which may limit or affect the flow or movement of some orall of particles 300. Insufficient headspace 223 may result in anincrease in pressure due to packing of particles 300, which may causeclogging or a reduction in the flow rate of nutritional formula 110during use. In other embodiments, particles 300 may not swell whensuspended in nutritional formula 110. In some embodiments, it may not benecessary for particles 300 to move as much when exposed to nutritionalformula 110. In such situations, chamber 222 may not include headspace223, or may include less of a headspace, if substantially no swelling ofparticles 300 occurs. In some embodiments, particles 300 may be prone toswelling, and chamber 222 may have a predetermined volume of headspace223 that becomes substantially filled or partially filled upon swellingof particles 300 during use. In such embodiments, sufficient headspace223 may be incorporated to allow room for particles 300 to swell whenwetted and to allow for sufficient space between particles 300 to allowroom for nutritional formula 110 to flow through particles 300 duringuse.

Preliminary experimentation has demonstrated that overfilling chamber222 with particles 300—and not leaving enough headspace 223—may resultin clogging or flow obstruction when particles 300 swell and packagainst each other. In an exemplary test run, 1.2 g of particles 300 wasfilled into a 3.70 mL chamber 222 having an interior diameter ofapproximately 1.56 cm, a height of approximately 1.94 cm, and a volumeof approximately 3.70 mL. This left a headspace 223 of approximately1/32 to approximately 2/32 inches above particles 300 in chamber 222. Asemi-elemental nutritional formula was flowed through device 200 at apump setting of 120 mL/hr. Under these conditions, approximately 500 mLof nutritional formula would be expected to be delivered withinapproximately 4 hrs. and 10 min. At the 1.2 g fill level, asignificantly slower flow rate was observed through device 200. In thenext runs, the fill weight was reduced to 1.1 g of particles 300 andthen 1.0 g of particles 300, incrementally increasing the amount ofheadspace 223 in chamber 222 to approximately 3/32 inches and toapproximately 4/32 inches, respectively. Two runs were performed at eachfill level. The results are show below. Reducing the amount of particles300 in device 200 from 1.2 g to 1.1 g or 1.0 g (providing slightly moreheadspace) yielded flow rates that were more in line with the expectedflow rates, based on the pump setting, indicating reduced or eliminatedflow obstruction. The reduced amount of particles 300 did not appear toimpact effectiveness.

TABLE 0 Particle fill amount and run time Particle fill amount Run time1.2 g 4 hr 32 min 1.2 g 5 hr 54 min 1.1 g 4 hr 2 min 1.1 g 4 hr 0 min1.0 g 4 hr 3 min 1.0 g 4 hr 12 min

Although the fill-level test described above refers to particle fillamount in terms of weight and describes an amount of headspace providedwhen chamber 222 is filled with a certain weight of particles, it isunderstood that if the size of chamber 222 is changed, or if a differentsize or density of particle is used, then filling chamber 222 with theexemplary weight of particles may yield a different amount of headspace.Headspace depends on the size and volume of the chamber and the size,type, and amount of particles.

The ratio of particle fill weight and headspace also depends on thedensity of the particles. While direct measurement and observation ofheadspace amount may be used to fill chamber 222 of device 200, use ofweight to fill device 200 may reduce fill variability that may be causedby static on particles 300. Static may cause particles 300 to initiallytake up more room in chamber 222, but, after particles 300 are allowedto settle and the static is allowed to dissipate, particles 300 maycompress and take up less space in chamber 222, ultimately providingmore headspace 223 than was intended upon initial visual observation ofheadspace 223. Using weight to assess fill level may, in someembodiments, help to control for the presence of static. Additionally oralternatively, static-removing measures may be utilized on particles 300prior to filling.

As alluded to above, however, under-filling devices 200, and leaving toomuch headspace 223, may result in decreased fat hydrolysis. In apreliminary fill-level test, devices 200 were filled with variousamounts of particles 300, ranging from 1.1 g to 0.6 g. The percenthydrolysis for the 1.1 g fill level was calibrated to 100%. The 0.8 gand 0.6 g fill levels displayed decreased hydrolysis of 77% and 65%,respectively, relative to the 1.1 g fill level, which was set at 100%.

As discussed above, the content of the solution to which particles 300are exposed may affect the swelling of particles 300. Therefore,particles 300 may swell by different amounts, depending on the type andcontent of the nutritional formula 110 to which particles 300 areexposed. A preliminary study was conducted to assess the swelling ofexemplary particles 300 upon exposure to water, to ethanol, and to twodifferent nutritional formulas, Peptamen®, a product of Nestle, andTwoCal® HN, a product of Abbott. In this experiment, approximately 10 to20 particles 300 were placed onto each of 4 different 100 μm meshfilters. The sample of particles 300 on each filter was measured under amicroscope in both the X and Y directions to determine the size of eachof the particles 300 in a given sample in a dry state. Then, thefilters, with the respective particle sample still on each of them, werecarefully placed into their own filter housings. Each of the filters andrespective particles was exposed to one of the 4 solutions (water,ethanol, Peptamen®, or TwoCal®). Each solution was pumped through therespective filter for 30 minutes at a pump flow rate of 120 mL/hr. After30 minutes of exposure, the filters with the respective particles (stillon top of the filters) were placed back under the microscope, and eachparticle on each filter was again measured in both the X and Ydirections to determine the size of the particles in the wetted state.Due to the shifting of the particles on the filters during theexperiment, the swelling of any individual particle was not tracked.Instead, each particle of the sample of 10-20 particles on each filterwas measured before and after exposure to the respective solution, andthe average measurements of each particle in a sample before and afterwetting were compared for each particle sample. The results are shownbelow in Table 1. As is demonstrated, different solutions (or differentnutritional formulas) may cause different amounts of swelling to occur,even when the same particle type is used.

TABLE 1 Swelling of particles after exposure to solutions Before Afterwetting wetting Average Wetting (average (average delta agent size μm)size μm) (μm) % Difference Water 197 212 15 8 TwoCal HN ® 188 210 22 12Peptamen ® 192 217 25 13 Ethanol 171 196 25 15

In some embodiments, particles 300 that swell at or below a certainthreshold amount may be used in device 200. For example, particles 300may be selected that have a percent difference between their dry stateand their wetted state of 15% or less, 20% or less, 25% or less, or 30%or less.

In some embodiments, device 200 may be filled with particles 300 so asto accommodate variable amounts of swelling that may occur whendifferent types of nutritional formulas 110 are used with device 200.For example, the fill level, and thus headspace 223, of chamber 222 maybe determined based on the amount of swelling that would occur whenparticles 300 are exposed to a nutritional formula that causes a maximumaverage amount of swelling of particles 300, compared to other types ofnutritional formulas. In such embodiments, the amount of particles 300or headspace 223 provided may accommodate even this maximum amount ofswelling. In other embodiments, chamber 222 may be filled with an amountof particles 300 that provides an amount of headspace 223 to accommodateuse with a particular nutritional formula 110 or a particular categoryof nutritional formulas. That particular nutritional formula 110 orparticular category of nutritional formulas may comprise a type ofsolvent that causes a certain amount of swelling in particles 300, andthus a device 200 tailored for use with this nutritional formula orcategory of formulas may include an amount of particles 300 and/orheadspace 223 able to accommodate the range of swelling that typicallyoccurs with that particular formula or category of formulas. In suchembodiments, the device 200 may be packaged with instructions for usewith that particular formula or category of formulas. Or, the device maybe sold with that particular nutritional formula.

The absolute number of particles 300 in chamber 222 may depend on thediameters, shapes, and size distributions of particles 300 and thevolume of chamber 222. In some embodiments, space may exist betweenparticles 300 and particles 300 may be less tightly packed, or, in someembodiments, less space may exist between particles 300 and particles300 may be closer together. For example, spherical particles 300, whenplaced together, may have spaces between adjacent particles, and thusparticles 300 may not take up all of the space in chamber 222 or thespace in chamber 222 available after accounting for headspace 223. Forexample, the total volume of particles 300 may take up from about 50% toabout 100%, from about 90% to about 95%, from about 85% to about 95%,from about 85% to about 90%, from about 80% to about 85%, from about 70%to about 80%, from about 60% to about 70%, from about 50% to about 60%,from about 80% to about 95%, from about 70% to about 95%, from about 60%to about 95%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, or from about 90% to about 100% ofthe space in chamber 222. In some embodiments, the number of particles300 in chamber 220 may range from about 10,000/mL to about 25,000/mL,from about 25,000/mL to about 50,000/mL, from about 50,000/mL to about75,000/mL, from about 75,000/mL to about 100,000/mL, from about100,000/mL to about 200,000/mL, from about 200,000/mL to about300,000/mL, from about 300,000/mL to about 400,000/mL, from about400,000/mL to about 500,000/mL, from about 500,000/mL to about600,000/mL, from about 600,000/mL to about 700,000/mL, from about700,000/mL to about 800,000/mL, from about 800,000/mL to about900,000/mL, or from about 10,000/mL to about 1,000,000/mL, depending onthe particle size and distribution.

In some embodiments, particles 300 in device 200 may be made up ofdifferent groups of particles having different median or mean diameters,and chamber 222 may contain different numbers of particles 300 from eachsize group. In some embodiments, different groups of particles havingdifferent median or mean diameters may be mixed together and/ordistributed randomly in chamber 222. In other embodiments, particles 300of different size groups may be substantially separated in layers, atleast in a dry state prior to use.

In some embodiments, the mass density of individual particles 300 may ormay not vary. The mass density of particles 300 may be adjusted byadjusting the materials forming particles 300, by modifying the monomercomponents of the copolymer of particles 300, by adjusting the size anddiameters of the pores and/or channels of particles 300, and/or byintroducing voids, pores, or a hollow core in particles 300. In someembodiments, particles 300 may have different mass densities. In someembodiments, if device 200 is placed in a vertical position with outlet230 or outlet 282 pointing down, when nutritional formula 110 flowsthrough chamber 222, particles 300 having a larger mass density thannutritional formula 110 may tend to flow or move towards outlet filter260 and particles 300 having a smaller mass density than nutritionalformula 110 may tend to float or move towards inlet filter 250. In someembodiments, particles 300 with a larger mass density than nutritionalformula 110 may collect along outlet filter 260 and may clog some of thepores of outlet filter 260. In some embodiments, particles 300 with asmaller mass density than nutritional formula 110 may collect alonginlet filter 250 and may clog some of the pores of inlet filter 250.

In some embodiments, the mass density of particles 300 may be selectedto more closely match the density of nutritional formula 110 such thatparticles 300 may be dispersed or suspended in nutritional formula 110,and may move more with the flow dynamics of nutritional formula 110. Insome embodiments, particles 300 may be dispersed or suspended innutritional formula 110 and may move more with the flow dynamics ofnutritional formula 110, depending on the orientation of device 200.This may decrease the propensity of particles 300 to collect at inletfilter 250 or outlet filter 260 and may promote more a centralized ordispersed distribution of particles.

In some embodiments, different groups of particles 300 having differentmedian or mean diameters may have different mass densities. This mayreduce the concern for filter clogging by promoting a more-disperseddistribution of particles in nutritional formula 110. In someembodiments, particles 300 may be divided into one or more groups havingan average mass density that substantially equals that of nutritionalformula 110, that are less than that of nutritional formula 110, andthat are more than that of nutritional formula 110. In some embodiments,particles 300 of about the same size, or having about the same median ormean diameters, or whose diameters follow the same distribution, mayhave about the same or may have different mass densities. In someembodiments, the mass density of particles 300 in chamber 222 may rangefrom about 0.25 g/mL to about 0.36 g/mL, from about 0.25 g/mL to about0.5 g/mL, from about 0.5 g/mL to about 0.8 g/mL, from about 0.8 g/mL toabout 1.0 g/mL, or from about 1.0 g/mL to about 1.5 g/mL, for example.

As shown in FIG. 7A, the surface of an individual particle 310 may begenerally smooth. In another embodiment, as shown in FIG. 7B, thesurface of a particle 310 may be uneven, irregular, or textured and mayinclude, for example, grooves, channels, indents, projections, and/orpores. In some embodiments, the depth and/or diameters of the poresand/or grooves on the surface of particle 310 may range from about 1 nmto about 10 nm, from about 10 nm to about 50 nm, from about 50 nm toabout 100 nm, from about 100 nm to about 250 nm, from about 250 nm toabout 500 nm, from about 500 nm to about 1 μm, from about 1 μm to about5 μm, from about 5 μm to about 10 μm, from about 10 μm to about 20 μm,from about 20 μm to about 30 μm, from about 30 μm to about 40 μm, fromabout 40 μm to about 50 μm, from about 10 nm to about 50 μm, or fromabout 1 nm to about 50 μm. In some embodiments, the grooves and/or poresof particle 310 may form any random geometric shape, may be irregularlydistributed on the surface, may be regularly shaped, and/or may beregularly distributed. In some embodiments, the size of the groovesand/or pores may depend on the composition of the polymeric material ofparticle 310. A non-smooth particle 310 will have a larger surface areathan a smooth particle 310 of the same shape having the same diameter.

In some embodiments, particles 300 in chamber 222 may include aplurality of particles 310 shown in FIG. 7A. In some embodiments,particles 300 in chamber 222 may include a plurality of particles 310shown in FIG. 7B. In other embodiments, particles 300 in chamber 222 mayinclude a mixture of particles 310 shown in FIG. 7A and particles 310shown in FIG. 7B.

FIGS. 7C and 7D show exemplary cross-sections of particle 310. In someembodiments, the interior of particle 310 may be generally compact orsolid, as shown in FIG. 7C. As shown in FIG. 7D, in some embodiments,the interior of particle 310 may be porous and may have nanoscopic,microscopic, and/or macroscopic structures, such as, for example, poresand/or channels. In some embodiments, the pores and/or cross-sections ofthe channels may be, for example, generally circular, elliptical, orirregular in geometric shape. In some embodiments, the pores and/orchannels may have network-type morphologies that can be eitherdisordered or assembled into ordered arrays. In some embodiments, thesurface of the pores and/or channels may be uneven, irregular, ortextured, and may be similar to the exterior surface of particle 310. Insome embodiments, the dimensions, such as the diameters and/orperimeters, of the pores and/or channels may vary along the length ofthe pores and/or channels, and may vary depending on the composition ofparticle 310 and/or the environment particle 310 is suspended in. Insome embodiments, the dimensions, such as the diameters and/orperimeters of the pores or channels, may increase or decrease whenparticle 310 is suspended in a solvent, such as nutritional formula 110.In some embodiments, the pores and/or channels of particle 310 may ormay not connect with the surface of particle 310, may extend throughparticle 310 from surface to surface, or may extend for discrete lengthswithin particle 310.

For a porous particle 310, as shown in FIG. 7D, the overall surface areaof particle 310 may be increased by the presence of internal poresand/or channels and may depend in part on the sizes, such as thediameters and/or perimeters, of the pores and/or channels. In someembodiments, the diameters of the pores and/or cross-sections of thechannels in particle 310 may range from about 1 nm to about 10 nm, fromabout 10 nm to about 50 nm, from about 50 nm to about 100 nm, from about100 nm to about 250 nm, from about 250 nm to about 500 nm, from about500 nm to about 1 μm, from about 1 μm to about 5 μm, from about 5 μm toabout 10 μm, from about 10 μm to about 20 μm, from about 20 μm to about30 μm, from about 30 μm to about 40 μm, from about 40 μm to about 50 μm,from about 10 nm to about 50 μm, or from about 1 nm to about 50 μm. Insome embodiments, the sizes of the pores and/or channels of particle 310may be substantially the same or may vary.

As described herein, reference to the surface of particle 310 may referto the outside surface of particle 310 and/or the internal surface ofthe pores and/or channels inside of particle 310. Also, reference to thesurface area of particle 310 may refer to the surface area of theoutside surface of particle 310 and/or the surface area of the poresand/or channels inside of particle 310 fluidly connected to the outsidesurface.

The characteristics of various embodiments of particles discussed abovemay be combined in any suitable manner. In some embodiments, as shownFIG. 7C, particle 310 may have a smooth outside surface and a solidcore. In some embodiments, as shown FIG. 7D, particle 310 may have asmooth outside surface and a porous core. In some embodiments, as shownFIG. 7E, particle 310 may have an uneven, irregular surface and acompact or solid core. In some embodiments, as shown FIG. 7F, particle310 may have an uneven, irregular outside surface and a porous core. Thecombination of an uneven surface and a porous core may provide increasedsurface area for particle 310 of a given size or diameter compared toits smooth and/or solid counterpart.

FIG. 8A is a scanning electron microscope image showing an exemplaryembodiment of particle 310 magnified 500 times. The scale bar of theimage is 10 μm. As shown in FIG. 8A, particle 310 has an uneven outsidesurface that has varied roughness at different locations. FIG. 8B is ascanning electron microscope image showing a cross-section of anexemplary embodiment of particle 310 magnified 500 times. The scale barof the image is 20 μm. As shown in FIG. 8B, particle 310 has microscopicpores of varied sizes throughout its interior. FIG. 9 further shows thepores of an exemplary particle 310. FIG. 9 is a scanning electronmicroscope image showing an exemplary embodiment of the pores andchannels inside of particle 310 magnified 50,000 times. The scale bar ofthe image is 100 nm. As shown in FIG. 9, the pores and channels insideof particle 310 have irregular sizes and shapes that vary at differentlocations.

In some embodiments, particles 300 may comprise one or more types ofparticle 310, for example, selected from one or more of the individualparticles 310 shown in FIGS. 7A-7F, FIG. 8, and/or FIG. 9, or asdiscussed above. Different types of individual particles 310 mayconstitute different numbers of all particles 300 in a given device 200,and may have different distributions of roughness, smoothness, porosity,diameters, materials, densities, and/or swelling properties.

As discussed above, particles 300 contained in device 200 have lipaseimmobilized on their surfaces. Lipase may be immobilized on exteriorsurfaces of particles 300, interior surfaces of particles 300, or acombination of exterior and interior surfaces. In some embodiments,functional groups of monomers of the polymeric material of particle 310may be present on the surface of particle 310 in order to bind lipase toparticles 300. Porous particle 310 having inside structures, such aspores and/or channels, may include functional groups located on both theoutside surface of particle 310 and the inside surface of the poresand/or channels. For example, the epoxy group of the monomer GMA of acopolymer material of particle 310 may be present on the surface ofparticle 310. In some embodiments, the epoxy groups may make up fromabout 0.01% to about 0.1%, from about 0.1% to about 1%, from about 1% toabout 2%, from about 2% to about 5%, from about 5% to about 8%, fromabout 8% to about 10%, from about 10% to about 15%, from about 15% toabout 20%, from about 0.01% to about 10%, from about 0.01% to about 15%,from about 0.01% to about 20% of the overall composition of polymericparticle 310 by weight. In some embodiments, the epoxy groups may belocated on the outside surface of particle 310. In some embodiments, theepoxy groups may be located on the inside surface of the pores and/orchannels or, in some embodiments, both the inside and outside surfacesof particle 310 may include epoxy groups or neither surface may includeepoxy groups. In some embodiments, the surface density or concentrationof epoxy groups on the outside surface of particle 310 may be higher orlower than that on the inside surface of the pores and/or channels ofparticle 310. In some embodiments, the amount of epoxy groups on thesurface of particle 310 may be capped to limit binding of lipase to anexcessive degree.

In some embodiments, the functional groups located on the surface ofparticle 310 may be used to adsorb or to bind to biomolecules orchemical molecules. In some embodiments, lipase 710 that hydrolyzesfats, including long-chain triglycerides and/or long-chain esters, forexample, triglycerides having LC-PUFAs, in nutritional formula 110, maybe attached or immobilized to the surface of particle 310 by covalentbinding. FIG. 10A shows an exemplary schematic of the crystal structureof lipase 710. FIGS. 10B-10C show exemplary schematics of the attachmentof lipase 710 to particle 310. As shown in FIG. 10B, particle 310 mayhave functional groups on its surface and may function as a carrier oflipase 710. Lipase 710, as shown in FIG. 10C, may covalently bind to thefunctional groups on the surface of particle 310 in a solution,resulting in a layer of lipase 710 on the surface of particle 310.Additionally, as shown in FIG. 10D, a certain number of individualparticles 310 having lipase 710 covalently bound to their surfaces makeup particles 300 in device 200.

As known in the art and described herein, cross-linking may refer to achemical bond that links one polymer chain to another. The chemical bondcan be a covalent bond or an ionic bond. In some fields, cross-linkingmay also refer to the use of a chemical linker to link proteinstogether. As used herein, “covalent bond” and “covalent binding” referto a stable, permanent or semi-permanent, irreversible, and/orcovalent-like bond for the attachment of lipase 710 to particle 310.

The embodiments of the present disclosure allow lipase 710 immobilizedby covalent binding to hydrolyze triglycerides or fatty acid esters innutritional formula 110 as nutritional formula 110 flows through device200 directly before ingestion by a subject. By covalently binding lipase710 to particles 300 and including one or more filters, device 200 isconfigured so that only a small amount of lipase 710 or substantially nolipase 710 may be included in the nutritional formula 110 ingested bythe subject. Although covalent binding is the primary way in whichlipase 710 is immobilized on particles 300, it is possible that duringthe immobilization process, background levels of adsorption may occur.Thus, in some embodiments, lipase 710 may not be solely immobilized bycovalent binding. Particles 300 with adsorbed lipase 710 may have lowerhydrolysis activity than covalently bound lipase 710 on particles 300.

During the research of the present disclosure, adsorption was testedinitially for attaching lipase 710 to particle 310. This is becauseadsorption is traditionally used for protein immobilization and worksvia hydrophobic forces. It is a simple and inexpensive means ofimmobilization. However, when adsorption was initially used forattaching lipase 710 to particles 300 when developing device 200,adsorption did not produce particles capable of effectively hydrolyzingfats in nutritional formula 110. After this initial testing, theinventors looked for other ways to immobilize or attach lipase 710 toparticles 300. As has been noted in previous publications, attachinglipase 710 to particle 310 via covalent binding may reduce or limit theenzymatic activity of lipase 710. For example, the enzymatic activity oflipase 710 may be reduced when covalently bound to particle 310 comparedto the enzymatic activity of lipase 710 in a soluble state. Thus, it wasinitially hypothesized that the enzymatic activity of lipase 710attached to particle 310 by covalent binding may be less than that oflipase 710 attached to particle 310 by adsorption. Yet, the enzymaticactivity of lipase 710 attached to particle 310 by covalent binding wasgreater than that of lipase 710 attached to particle 310 by adsorption.Further, adsorption did not achieve higher performance or efficiency forhydrolyzing fats in nutritional formula 110 when compared to covalentbinding. Example 1, described below, compares the enzymatic activitiesof lipase 710 immobilized to particles 300 by adsorption and by covalentbinding and suggests that lipase 710 immobilized to particles 300 bycovalent binding has greater enzymatic activity, better performance inhydrolyzing fats, and less release of lipase 710 into nutritionalformula 110.

Example 1: Comparison of the Immobilization of Exemplary Lipase 710 toExemplary Particles 300 Using Adsorption and Using Covalent Binding

A total of six test samples of exemplary lipase 710 attached toexemplary particles 300 were prepared. Three test samples, hereinreferred to as A1, A2, and A3, were prepared by adsorption of exemplarylipase 710 to particles 300 while the other three test samples, hereinreferred to as C1, C2, and C3, were prepared by covalent attachment ofexemplary lipase 710 to particles 300. Exemplary particles 300 forsamples A1, A2, and A3 were formed from styrene (A1, A2) or methacrylate(A3) polymer with no reactive groups. Exemplary particles 300 forsamples C1, C2, and C3 were formed from methacrylate polymer withreactive (epoxy) groups for covalent bonding. All six test samples wereprepared with 125 mg of lipase 710 per gram of particles 300. Covalentattachment of lipase 710 to particles 300 for samples C1, C2, and C3 wasachieved by allowing lipase 710 to covalently bind to the epoxy groupson the surface of particles 300. The diameters of particles 300 rangedfrom 220 μm to 500 μm, and particles 300 were coated with PEG. Threeassays were performed to evaluate the six test samples and to comparethe immobilization of lipase 710 to particles 300 using adsorptionversus using covalent binding.

First, a titration assay was performed for each test sample to evaluatethe potency or specific activity of lipase 710 attached to particles 300in each sample against an emulsified raw fish oil substrate having 40%DHA triglycerides by weight. Second, a lipase release assay wasperformed to assess the amount of lipase 710 released from particles 300of each test sample. Third, a fat hydrolysis performance assay wasperformed to test the fat hydrolysis performance of lipase 710 attachedto particles 300 in each sample in an exemplary device 200. The resultsare discussed in the following.

In the titration assay, for each particle test sample, 12 mg of dryparticles 300 were added to an emulsified fish oil substrate. Thesubstrate was equilibrated to 37° C. with stirring. The specificactivity of lipase 710 attached to particles 300 was measured in eachtest sample. The specific activity is defined as the amount of freefatty acids generated per gram of the total lipase 710 attached toparticles 300 in a given amount of time under the assay conditions. Theamount of free fatty acids generated by each sample was measured bytitrating the fish oil substrate with NaOH solution to keep the fish oilsubstrate at a constant pH. During the hydrolysis reaction, as theimmobilized lipase 710 hydrolyzed triglycerides in the raw fish oilsubstrate, free fatty acids were generated, and NaOH solution was addedto neutralize the acids. The moles of NaOH added during the reaction toneutralize the acid equaled the moles of free fatty acids produced bylipase 710 in each sample.

As shown in Table 2 and FIG. 11, the test samples of lipase 710 attachedto particles 300 via covalent binding, i.e., C1, C2, and C3, generallyhad higher specific activities than the test samples of lipase 710attached to particles 300 via adsorption, i.e., A1, A2, and A3. Thisresult was unexpected from previous publications regardingimmobilization using adsorption and using covalent binding. Withoutbeing bound to this theory, it is hypothesized that this surprisingresult may be due to the fact that adsorption is a type of nonspecificbinding mechanism that may cause the active site of lipase to beattached to the surface of a particle, reducing the accessibility of theactive site of lipase to the fat molecules in the DHA oil substrate. Thereduced accessibility may reduce the overall activity of lipase 710immobilized on particles 300.

TABLE 2 Specific activities of test samples including lipase 710immobilized to particles 300 via adsorption and covalent binding Testsample Immobilization mode Specific activity (U/g) A1 Adsorption 691 A2Adsorption 69 A3 Adsorption 24 C1 Covalent binding 350 C2 Covalentbinding 938 C3 Covalent binding 1950

In the lipase release assay, 1 g of each test sample was suspended in 10mL distilled water in a centrifuge tube. Each centrifuge tube wasrotated end over end using an automatic shaker at room temperature forabout 12 hours. At 1-hour, 3-hour, and 12-hour time points, each testsample was centrifuged, and a measurement sample from the supernatant ofeach test sample was collected to obtain a concentration of lipase 710that had detached from particles 300 at those time points. Theconcentrations of lipase 710 in the measurement samples were quantifiedby measuring the absorbance of the measurement samples at a wavelengthof 280 nm using a spectrophotometer. As shown in FIG. 12, at all of thetime points, the test samples having lipase 710 immobilized to particles300 via adsorption, A1, A2, and A3, had higher concentrations of lipase710 in the supernatant than the test samples having lipase 710immobilized to particles 300 via covalent binding, C1, C2, and C3.Therefore, the results show that the attachment of lipase 710 toparticles 300 by covalent binding was stronger and more stable thanattachment of lipase 710 to particles 300 by adsorption.

In the fat hydrolysis assay, an exemplary nutritional formula 110,Peptamen AF®, was used. One gram of each test sample was placed in anexemplary device 200. Each exemplary device 200 had a body 210 made ofclear polycarbonate and an inlet filter 250 and an outlet filter 260made using 3-D printing methods. For each exemplary device 200, thediameter of inlet 242 was approximately 6.6 mm; the diameter of inletfilter chamber 214 tapered from 2.6 mm to 15 mm; the diameter of inletfilter 250 was approximately 15 mm and the thickness of inlet filter 250was approximately 3.2 mm; the diameter of chamber 222 tapered from 15 mmto 17 mm; the diameter of outlet filter chamber 224 was approximately 17mm; the diameter of outlet filter 260 was approximately 17 mm and thethickness of outlet filter 260 was approximately 3.2 mm; the diameter ofoutlet 230 was approximately 17 mm; the interior diameters of inlet 272and inlet chamber 274 were approximately 15 mm; the exterior diametersof inlet 272 and inlet chamber 274 were approximately 17 mm; and thediameter of outlet 282 was approximately 2 mm. Inlet filter 250 andoutlet filter 260 were made from polyethylene and had an approximateporosity of 100 μm. Each device 200 was filled with about 1 g to about1.2 g of particles 300, leaving a headspace 223 of approximately 1 mmabove particles 300 in a dry condition.

Peptamen AF® solution was driven through device 200 by an exemplary pump120 at a set flow rate of 2 mL/min. As Peptamen AF® solution passedthrough each test sample in each device 200, the fat, such astriglycerides, in the Peptamen AF® solution was hydrolyzed by lipase 710attached to particles 300 of each test sample. During the flow of thePeptamen AF® solution through device 200, for each test sample, threemeasurement samples were collected. One sample was collected at t₀before Peptamen AF® solution was exposed to particles 300, one samplewas collected at t_(i) just as the Peptamen AF® solution began flowingout of device 200, and one sample was collected at t₃₀ 30 minutes aftert_(i). The amount of free fatty acids in each measurement samplecollected at each time point was measured using a quantitativecolorimetric assay (Abcam® Free Fatty Acid Quantification Kit).Performance of fat hydrolysis by each test sample in device 200 wasevaluated based on the amount of free fatty acids generated. FIG. 13shows the amount of free fatty acids generated by the test samplesplaced in device 200. The results show that when nutritional formula 110was flowed though lipase 710 immobilized to particles 300 in device 200,lipase 710 immobilized to particles 300 using covalent binding (C1, C2,and C3) had better performance in hydrolyzing fats in nutritionalformula 110 than lipase 710 immobilized to particles 300 usingadsorption (A1, A2, and A3).

As shown in Example 1, an advantage of using covalent binding of lipase710 to particle 310 is the strength of the bond, i.e., the stabilityand/or strength of the immobilization. Comparatively speaking,adsorption is reversible and has the disadvantage of incompleteattachment, which may allow lipase to detach from a particle. Thisdisadvantage may allow a substantial amount of lipase to mix with anutritional formula, and if used, may be delivered to the patient as thenutritional formula flows through device 200. This may be undesirable tosubjects in need of the fatty nutrients in nutritional formula 110, suchas infant populations or immune compromised patients, because excesslipase may negatively affect their GI tracts, as discussed previously.

By contrast, with covalent binding, at least one covalent bond formsbetween a support material and a functional group on an amino acid onthe surface of the lipase. The functional groups that may bind thelipase to the support material include, e.g., amino, carboxyl,sulfhydryl, hydroxyl, imidazole, or phenolic groups, and are notessential for the catalytic activity of the lipase. In some embodiments,the amino groups of the side chains of one or more lysine residues oflipase 710 may react with the epoxy groups on the surface of particle310 and form covalent bonds. In order to protect the active site,immobilization can be carried out in the presence of a substrate or acompetitive inhibitor. For an example of lipase immobilized by covalentbinding, see S. Emi et al., European Polymer Journal 30(5):589-595(1994). Supports suitable for covalent binding may include, e.g.,Immobead™ (ChiralVision).

As noted above, covalent binding is a stronger and/or a more stable typeof interaction between lipase 710 and particle 310, which may result instronger and/or irreversible binding and reduced detachment of lipase710 from particles 300. Thus, covalent binding of lipase to particles300 is used in embodiments of the present disclosure, to reduce oreliminate the amount of lipase 710 that may detach from particles 300 asnutritional formula 110 flows through chamber 222 (and is ultimatelydelivered to a subject). The covalent binding of lipase 710 to particles300 may advantageously improve the stability of the attachment, renderlipase 710 and particles 300 reusable in some embodiments if desired,and may allow nutritional formula 110 that has been hydrolyzed by lipase710 attached to particles 300 to have little or substantially nocontamination of lipase 710. Purified lipase 710 that is substantiallyfree from non-active lipase and/or non-lipase entities or has reducedamounts of non-active lipase and/or non-lipase entities may allow forimproved binding efficiency and hydrolysis efficiency due to improvedcovalent binding of lipase 710 on particles 300. That said, as mentionedabove, even with purified lipase 710, background levels of adsorptionmay occur during the process of covalently binding the lipase 710 toparticles 300, although covalent binding may be the predominant mode ofattaching lipase 710 to particles 300.

As described herein, hydrolysis efficiency may be used to describe theperformance of device 200 in hydrolyzing the fats (e.g., long-chainfatty acid triglycerides and/or long-chain fatty acid esters) innutritional formula 110. Hydrolysis efficiency may be defined as thepercentage of fat hydrolyzed out of the total amount of fat innutritional formula 110 after nutritional formula 110 has been flowedthrough device 200. In addition, lipase 710 used in the devices hereingenerally cleaves two out of three bonds in a triglyceride, i.e., at thesn-1 and sn-3 positions, leaving an sn-2 monoglyceride. Accordingly,100% hydrolysis is achieved when two out of three bonds are broken in agiven triglyceride. As described in more detail in the followingembodiments of the present disclosure, it is recognized that it may beadvantageous to maximize the exposure or interaction of lipase 710attached to particles 300 with the fat molecules in nutritional formula110 in chamber 222 to improve the hydrolysis efficiency of device 200 inorder to supply pre-hydrolyzed free fatty acids and monoglycerides fromnutritional formula 110 to a subject in a shorter period of time at thepoint of care to allow for more effective absorption of free fatty acidsand monoglycerides by the body, for example, into plasma and/or tissues.Reducing the exposure time may allow for a reduction in the amount oftime needed to provide nutritional formula 110 to a patient, which mayallow patients to avoid overnight feeding, if desired, withoutsignificantly effecting hydrolysis efficiency.

Lipases can be obtained from animals, plants, and from many natural orgenetically engineered microorganisms. Many commercially availablelipase products are derived from animals and are particularlysusceptible to degradation by digestive enzymes. A less frequently usedalternative is microbial lipase, i.e., lipase produced in bacteria orfungus, such as, e.g., yeast. Certain microbial lipases retain activityover a wider pH range than animal or plant lipases, thus eliminating theneed for enteric coated tablets. However, microbial enzymes tend to bedegraded by trypsin in the small intestine, thereby reducing theiravailability to breakdown triglycerides and esters in the gut. In someembodiments, lipases 710 used in the present disclosure includebacterial lipases, fungal lipases, or both. Microbial lipases may or maynot require a co-lipase or may or may not be affected by bile salts.

The specificity and kinetics of individual types of lipase can varysignificantly. Specificity of lipases is controlled by the molecularproperties of the enzyme, structure of the substrate, and factorsaffecting binding of the enzyme to the substrate. Types of specificityinclude substrate specificity. In some embodiments, lipase 710 is chosento selectively hydrolyze triglycerides and/or esters having at least onelong-chain and/or medium-chain polyunsaturated fatty acid. In someembodiments, similar to human pancreatic lipase, lipase 710 mayspecifically hydrolyze the ester bonds at positions 1 and 3 of theglycerol backbone of a triglyceride and generate two free fatty acidsand one monoglyceride from the triglyceride, as shown in FIG. 14. Insome embodiments, the polyunsaturated fatty acid generated by thehydrolysis of the triglyceride by lipase 710 may include one or more ofdocosahexaenoic acid (DHA), arachidonic acid (ARA), eicosapentaenoicacid (EPA), and linoleic acid (LA). In some embodiments, lipase 710 maybe selected based on assaying its affinity to hydrolyze one or moretypes of triglycerides having LCTs, such as LC-PUFAs.

It has now been determined that lipase produced by Chromobacteriumviscosum, Pseudomonas fluorescens, Burkholderia cepacia, and Rhizopusoryzae have greater specificity for DHA, EPA, and ARA than otherlipases, such as lipase produced by Candida rugosa, Rhizomucor miehei,Penicilium camemberti, Aspergillus niger, and Aspergiffis oryzae. Thus,lipase 710 may be a microbial lipase selected from at least one ofChromobacterium viscosum lipase, Pseudomonas fluorescens lipase,Burkholderia cepacia lipase, and/or Rhizopus oryzae lipase. In someembodiments, lipase 710 is Chromobacterium viscosum lipase, Pseudomonasfluorescens lipase, or Rhizopus oryzae lipase. In some embodiments,lipase 710 is Rhizopus oryzae lipase. In some embodiments, lipase 710has specific activities for triglycerides having DHA, EPA, and/or ARAthat are comparable to the specific activities of one or more ofChromobacterium viscosum lipase, Pseudomonas fluorescens lipase, orRhizopus oryzae lipase.

Reference to the lipase of certain species, such as Chromobacteriumviscosum lipase, Pseudomonas fluorescens lipase, Burkholderia cepacialipase, and Rhizopus oryzae lipase, does not necessarily mean that thelipase was prepared directly from the native host species. For example,the same lipase could be produced recombinantly in another host cell.

In some embodiments, the enzyme may be selected from at least one ofChromobacterium viscosum lipase, Pseudomonas fluorescens lipase,Rhizopus oryzae lipase, Thermomyces lanuginosus lipase, Pseudomonasfluorescens lipase, Bacillus subtilis lipase, Candida rugosa lipase,Mucor javanicus lipase, Lecitase, Rhizopus niveus lipase, Rhizomucormiehei lipase, Aspergillus niger lipase, Penicillium camemberti lipase,Burkholderia cepacia lipase, Aspergillus oryzae lipase, Pseudomonasstutzeri lipase, Alcaligenes spp. lipase, Candida antarctica lipase,Hansenula polymorpha lipase, Humicola insolens lipase, Thermomyceslangunosa phospholipase, lecithinase phospholipase, or a lipase orphospholipase from any recombinant species within any of the abovegenus, or any suitable lipase or phospholipase or combination thereof.

In some embodiments, at least one type of lipase 710 may be attached toan individual particle 310. In some embodiments, different types oflipase 710 may be attached to the same particle 310 or to differentgroups of particles that make up particles 300. Different groups ofparticles may have different lipases, different median or meandiameters, different surface areas, different functional groupconcentrations or types, and/or may be made with different types ofpolymeric material, such as solid or porous polymeric materials.

In some embodiments, lipase 710 may be an extract from a microbialpopulation, for example, Rhizopus oryzae, and may contain other proteinsor enzymes. In some embodiments, lipase 710 may comprise gastric lipase,and/or non-lipase enzymes, such as lecithinase. In some embodiments,lipase 710 may be purified before attachment to a particle 310, and/ormay be modified by adding functional chemical groups or chemicallinkers. In some embodiments, lipase 710 may hydrolyze more than onetype of fat, such as different triglycerides having one or moredifferent long-chain polyunsaturated fatty acids or phospholipids.

In some embodiments, lipase 710 may catalyze hydrolysis of fats ortriglycerides at a range of pH values and may have a maximum hydrolysisactivity at pH values ranging from about 5 to about 8. The pH of a givennutritional formula 110 may be around a neutral pH, such as from aboutpH 6 to pH 8, thus a lipase 710 may be selected that hydrolyzes fatsefficiently at substantially the same pH range as that of nutritionalformula 110. In some embodiments, a lipase 710 may be selected that hasa peak activity at the pH of nutritional formula 110. Unlike humanpancreatic lipase, lipase 710 may not need co-factors to hydrolyze fatsefficiently. In some embodiments, the enzymatic activity of lipase 710may not be affected by bile salts.

In some embodiments, lipase 710 may be active over temperatures rangingfrom about 4° C. to about 35° C. In order to prevent nutritional formula110 from spoilage, nutritional formula 110 may be stored andrefrigerated at a temperature ranging from 4° C. to about 10° C.Nutritional formula 110 may be delivered to the patient after beingretrieved from refrigerated storage or may be delivered after beingwarmed to room temperature, e.g., about 20° C. to about 25° C. Thus, thetemperature of nutritional formula 110 typically may range from about 4°C. to about 25° C. In some situations, nutritional formula 110 may bewarmed, for example, to body temperature, about 36° C. to about 37° C.,before delivery. In some embodiments, a lipase 710 may be selected thathydrolyzes fats efficiently at substantially the same temperature rangeas that of nutritional formula 110. Microbial lipases also generallyhave an optimal activity level at a certain pH or a certain pH range. Insome embodiments, lipase 710 may be suited for use with a neutral pH ofnutritional formula in addition to, or instead of, the lower pH range ofthe stomach environment. In some embodiments, lipase 710 may be lessactive in the gastrointestinal system, allowing for improved safety. Insome embodiments, a lipase 710 may be selected that has a peak activityat the temperature of nutritional formula 110 prior to delivery. In someembodiments, a lipase 710 may be selected that has sufficient activityover the range of temperatures that nutritional formula 110 may bedelivered at.

In some embodiments, the density of lipase 710 attached to particle 310may be controlled by adjusting the concentration of the functionalgroups, such as the epoxy groups, of the polymeric material of particle310. A decrease in the concentration of epoxy groups present on thesurface of particle 310 may decrease or limit the density of lipase 710attached to particle 310. In some embodiments, the density of lipase 710attached to particle 310 may range from about 10 mg to about 100 mg, 100mg to about 200 mg, from about 100 mg to about 300 mg, from about 100 mgto about 400 mg, from about 100 mg to about 500 mg, from about 200 mg toabout 300 mg, from about 200 mg to about 400 mg, from about 200 mg toabout 500 mg, from about 300 mg to about 400 mg, from about 300 mg toabout 500 mg, or from about 400 mg to about 500 mg per gram of polymericparticle 310.

In some embodiments, the density of lipase 710 attached to the surfaceof a given particle 310 may be increased to increase the amount oflipase 710 on particles 300 in device 200 to more efficiently hydrolyzefats, such as long-chain fatty acid triglycerides and/or long-chainfatty acid esters, in nutritional formula 110. In some embodiments,increasing the density of lipase 710 attached to the surfaces ofparticles 300 may allow fewer particles 300 to be used in device 200without decreasing the amount of lipase 710 in device 200, and thuspotentially without substantially affecting the overall hydrolysisefficiency of device 200. In some embodiments, however, increasing thedensity of lipase 710 attached to the surface of particles 300 may notincrease the overall efficiency of lipase 710 on particles 300 or mayreach a threshold level of efficiency. For example, although anincreased amount of lipase 710 may be bound to an individual particle310, lipase 710 may be immobilized on the surface of the pores and/orchannels inside of particle 310, and, if the sizes of the pores and/orchannels are smaller than the fat molecules to be hydrolyzed and/or aresubstantially hydrophilic, fat molecules in nutritional formula 110 maynot come into contact with the pores and/or channels and may not reactwith lipase 710 bound there. In such situations, increasing the amountof lipase 710 bound inside of particle 310 may not increase the overallhydrolysis efficiency of particles 300 or device 200.

In some embodiments, increasing the density of lipase 710 attached tothe surface of particle 310 beyond a threshold may not increase thehydrolysis efficiency of lipase 710 on particle 310 or may even decreasethe efficiency in some instances. For example, increasing the density oflipase 710 may affect the orientation of lipase 710 on particle 310 ormay increase the steric hindrance between adjacent lipase molecules onparticle 310, and/or may reduce the flexibility or accessibility oflipase 710 to the fat molecules in nutritional formula 110. If thisoccurs, then even through there is more lipase 710 on particle 310, thefats in nutritional formula 110 may not be able to interact with theactive site of the lipase, and adjacent lipase molecules may obstructeach other. In some embodiments, the density of lipase 710 attached tothe surface of particle 310 may be reduced to allow sufficientflexibility of lipase 710 and/or to reduce steric hindrance betweenadjacent lipases molecules on particle 310, and thus to preserve and/orincrease the overall activity of lipase 710 attached to particle 310 bymaking it accessible to the fats to be hydrolyzed. In some embodiments,if a threshold efficiency is reached, then an amount of lipasesubstantially equivalent to that threshold amount may be used, sinceincreasing the amount of lipase may only add cost with no substantialefficiency benefit.

In some embodiments, the purity of lipase 710 may be altered to increasethe covalent binding and hydrolysis efficiency of lipase 710 on particle310. For example, some lipase enzyme preparations may include proteinand polysaccharide carryover materials from their isolation orproduction, or they may contain diluents or inactive lipase. These othermaterials may interfere with the enzyme active sites of active lipase710, may compete for covalent binding sites on particles 300, maysterically hinder lipase 710, and/or may prevent the substrate fromreadily reaching the active site. In some embodiments, these non-activeand non-lipase entities may be removed from the enzyme preparationduring the process of immobilization, or, in some embodiments, thesenon-active and non-lipase entities may be removed from the enzymepreparation before immobilization. Removal of non-active and/ornon-lipase entities may provide for an increase in the overall activityof lipase 710 attached to particles 300. In some embodiments, the massratio of active lipase to enzyme preparation before immobilization maybe as low as 5% and as high as essentially 100%.

In some embodiments, the amount of lipase 710 attached to particle 310may be proportional to the surface area of particle 310. For example, ifa first particle 310 has a larger diameter, and thus a larger surfacearea than a second particle 310, then at an equal density of lipase 710,the first particle 310 will have a larger amount of lipase attached toit than the second particle 310. For particles of the same size, aparticle 310 having a porous core may have a larger surface area than aparticle 310 having a solid core, thus if the densities of lipase 710attached to the surface area of the particles are equal, then the amountof lipase 710 attached to the particle with a porous core may be greaterthan the particle 310 with a solid core. Similarly, for particles of thesame size, a particle 310 having an uneven, irregular surface may have alarger surface area than a particle 310 having a smooth surface, thus ifthe density of lipase 710 attached to the surface of the particles isthe same, the amount of lipase 710 attached to the particle 310 with anuneven, irregular surface may be more than that attached to the particle310 with a smooth surface. Therefore, particles 300 made up ofindividual particles 310 having a larger surface area, such as particles310 having uneven surfaces and porous cores, may provide a largeroverall surface area and thus a larger amount of lipase 710 thanparticles 300 made up of individual particles 310 having a smallersurface area and thus a smaller amount of lipase 710, such as particles310 having smooth surfaces and solid cores.

In some embodiments, the amount of lipase 710 in chamber 222 may beproportional to the total surface area of particles 300 contained inchamber 222. The surface area and volume of individual particles 300 isproportional to the size or diameter of that particle 310. The surfacearea and volume of a spherical particle having a diameter of D can becalculated as π*D² and (π*D³)/6, respectively. In some embodiments,since chamber 222 may have a predetermined volume, there will be amaximum number of particles 300 that can be placed in chamber 222. Forexample, if chamber 222 has a volume of V₀, particles 300 having amedian or mean diameter of D₁ that can be placed in chamber 222 beingN₁, and particles 300 having a median or mean diameter of D₂ that can beplaced in chamber 222 being N₂, where D₁ is larger than D₂, N₁ is thensmaller than N₂. In other words, for a given volume of chamber 222, thenumber of particles 300 having a larger diameter that are able to fit inchamber 222 will be less than the number of particles 300 having asmaller diameter. In this situation, the total surface area of particles300 is inversely proportional to the median or mean diameter ofparticles 300, if all other variables are equal. Thus, given a totalvolume of particles 300, the total surface area of particles 300 havinga larger median or mean diameter is less than the total surface area ofparticles 300 having a smaller median or mean diameter. Therefore, insome embodiments, to increase the surface area of particles 300, chamber222 of body 210 may be made in a larger volume to accommodate moreparticles 300. In other embodiments, to increase the surface area ofparticles 300, given a certain volume of chamber 222, the median or meandiameter and/or diameters of particles 300 may be selected to besmaller.

In some embodiments, chemical linkers may be used to link the surface ofparticle 310 to lipase 710. Such chemical linkers may increase thedistance between lipase 710 and the particle 310 to which it isattached. For example, a chemical linker may increase the distance oflipase 710 further away from the surface of particle 310 at a range fromabout 0.1 nm to about 1 nm, from about 1 nm to about 3 nm, from about 3nm to about 4 nm, from about 4 nm to about 6 nm, from about 6 nm toabout 8 nm, from about 8 nm to about 10 nm, from about 12 nm to about 14nm, from about 14 nm to about 16 nm, from about 16 nm to about 18 nm,from about 18 nm to about 20 nm, from about 0.1 nm to about 3 nm, fromabout 0.1 nm to about 4 nm, from about 0.1 nm to about 8 nm, from about0.1 nm to about 10 nm, from about 0.1 nm to about 12 nm, from about 0.1nm to about 14 nm, from about 0.1 nm to about 16 nm, from about 0.1 nmto about 18 nm, or from about 0.1 nm to about 20 nm. This may increasethe mobility or flexibility of lipase 710, reduce steric hindrance ofadjacent lipase molecules, and/or orient the active site of lipase 710to the fat molecules in nutritional formula 110, and thus may preserveor increase the enzymatic activity of lipase 710. In some embodiments,chemical linkers may allow lipase 710 to take a certain orientation onthe surface of particle 310 to orient the active site of lipase 710towards the fat molecules to be hydrolyzed in nutritional formula 110.In some embodiments, spacer molecules may be attached or chemicallylinked to the surface of particle 310 and may be placed between adjacentlipase molecules to reduce the steric hindrance among adjacent lipasemolecules on the surface of particle 310.

In some embodiments, different particles 300 may have a different amountand/or density of lipase 710 attached to their surfaces. As notedherein, particles 300 may include all types of individual particles 310as described above, or similar particle types may have different sizes,shapes, mass densities, and/or densities of immobilized lipase 710. Insome embodiments, each of these different particle types may have adifferent density of lipase 710, a different surface area, and/or adifferent amount of immobilized lipase 710, etc.

Increasing the overall surface area of particles 300 and/or the totalamount of lipase 710 in chamber 222 may increase the exposure to orinteraction between lipase 710 and the fat molecules in nutritionalformula 110, which may improve the efficiency of device 200 forhydrolyzing fats, such as long-chain polyunsaturated triglyceridesand/or long-chain polyunsaturated esters, in nutritional formula 110.

In some embodiments, the surface of an individual particle 310 may behydrophobic or partially hydrophobic. For example, the surface ofparticle 310 may be hydrophobic and thus may have limited wettingability or no wetted state. In some embodiments, the hydrophobic surfaceof particle 310 may attract fat molecules from an aqueous solution, anoil-water emulsion, or a complex nutritional liquid, such as nutritionalformula 110, through hydrophobic interactions. Such hydrophobicinteractions may increase the accessibility of the fat molecules tolipase 710 attached to particle 310 and may facilitate the hydrolysis ofthe fat molecules in nutritional formula 110 by lipase 710. In someembodiments, the surface of particle 310 may be hydrophilic or partiallyhydrophilic. For example, the surface of particle 310 may be hydrophilicand may be wetted upon suspension in an aqueous solution, an oil-waterliquid, and/or nutritional formula 110. In some embodiments, thepolymeric material of particle 310 may be partially hydrophilic andpartially hydrophobic, e.g., by including one or more polymers or acopolymer. In such embodiments, particle 310 may be both hydrophilic andhydrophobic on the surface, and may attract fat molecules in nutritionalformula 110 and may be wetted in nutritional formula 110. In someembodiments, the outside surface of particle 310 may be hydrophilic, andthe surface of the pores and/or channels inside particle 310 may behydrophobic, or vice versa.

Having a hydrophilic surface of particle 310 or wetting of particle 310may be beneficial for the enzymatic activity of lipase 710 attached tothe surface of particle 310. In some embodiments, as shown in FIG. 15,particle 310 may have a polyethylene glycol (PEG) coating 315. In someembodiments, PEG coating 315 on the outside surface of particle 310 mayimprove the wetting ability of particle 310 when particle 310 issuspended in a solvent including water, such as nutritional formula 110,thereby creating a wetted surface environment beneficial for theenzymatic activity of lipase 710. In some embodiments, PEG coating 315may improve the stability of the attachment of lipase 710 to particle310. In some embodiments, the amount of PEG coating 315 may range fromabout 0 to about 2%, from about 2% to about 5%, from about 5% to about8%, from about 8% to about 10%, from about 5% to about 10%, from about2% to about 10%, or from about 0 to about 10% of the overall compositionof particle 310 by weight. Alternatively, other coatings or combinationsof coatings may be used to improve the wetting ability of particle 310when particle 310 is suspended in a solvent including water. Alternativecoatings may include, e.g., a lecithin coating, a polyvynylpyrrolidonecoating, a polyvinyl alcohol coating, a non-ionic surfactant coating, analcohol coating, such as dodecanol, a glycerol coating, a propanediolcoating (e.g., 1,2-propanediol), water, or any suitable coating that mayimprove the wetting ability of particle 310 in nutritional formula 110.In some embodiments, wetting agents may be included in the coating ofparticle 310 to improve the wetting ability of particle 310 innutritional formula 110.

In some exemplary embodiments, PEG may be used to provide stability forimmobilization of enzymes. In some embodiments, the inclusion of 2% to10% PEG, by weight, has yielded shelf-life stability of lipase in device200 of at least approximately 18 months when stored at routine storageconditions (5° C.±3° C. and 25° C.±2° C. at 60% RH±5% RH). In someembodiments, the absence of or reduced levels of PEG on particles 300may also yield suitable shelf-life stability of lipase on particles 300.

In some embodiments, particle 310 may comprise a polymeric matrix and/orlattice. For example, the polymeric matrix may be made of a porouscopolymer having pores and/or channels, and lipase 710 may aggregate andbe entrapped in particle 310. In such situations, the active site of thelipase may remain exposed and interact with the fat molecules ormicelles. For example, when nutritional formula 110 is flowed throughchamber 222 and particles 300, fat molecules of nutritional formula 110may enter the complex matrix and/or lattice, e.g., by convection and/ordiffusion, and then mix with, interact with, or be hydrolyzed by lipase710 or aggregates of lipase 710 entrapped in the matrix and/or lattice.

As discussed previously, one or more filters may be used to retainparticles 300 in chamber 222, prevent clogging, and/or direct or affectthe flow of a liquid, including nutritional formula 110, through device200 and particles 300. Inlet filter 250 and outlet filer 260 may includea mesh 800 having an intake surface 810 and an outtake surface 820, asshown in FIGS. 16A and 16B. FIG. 16A shows a cross-section of anexemplary embodiment of mesh 800. As shown in FIG. 16A, in someembodiments, mesh 800 may be a traditional, screen-type mesh 800 havinggenerally ordered channels for passing fluid. Such channels may bepatterned, for example, straight, as a comb, e.g., a honeycomb, and/orradially distributed. For example, as shown in FIG. 16A, mesh 800 mayhave straight paths in its structure to allow nutritional formula 110 topass through. In some embodiments, nutritional formula 110 may be flowedthrough the straight paths of mesh 800 directed by pump 120, by gravityfeeding, or via use of a syringe. The diameters and/or relativepositions of the straight paths may be uniform or may vary across mesh800.

Mesh 800 may impose hydraulic resistance to the flow of nutritionalformula 110, and the magnitude of the hydraulic resistance and the flowof nutritional formula 110 may depend on the diameters and/or locationsof the paths of mesh 800. For example, if the diameters or perimeters ofthe paths of mesh 800 are sufficiently large, nutritional formula 110may be met with a small magnitude of hydraulic resistance, and may passthrough the paths near the middle of mesh 800 more than the paths at theperipheral of mesh 800, resulting in a more focused flow of nutritionalformula 110 at an outtake surface 820 of mesh 800. If the diameters ofthe paths of mesh 800 are sufficiently small and/or the paths areoriented in a manner to distribute flow, nutritional formula 110 may bemet with a larger magnitude of hydraulic resistance, and may thus bedistributed at intake surface 810 of mesh 800 and may pass more evenlythrough the paths across mesh 800, resulting in a more distributed flowof nutritional formula 110 at outtake surface 820 of mesh 800. In someembodiments, at least some of the paths of mesh 800 may be angledoutwards toward the periphery of mesh 800, directing the flow ofnutritional formula 110 to the periphery of chamber 222 and allowingnutritional formula 110 to be distributed across particles 300 inchamber 222. Mesh 800 that provides a more distributed flow ofnutritional formula 110 may allow nutritional formula 110 to be exposedto more particles 300, and thus more lipase 710 in chamber 222,potentially increasing the efficiency of device 200 for hydrolyzing fatsin nutritional formula 110.

In some embodiments, as shown in FIG. 16B, mesh 800 may be a porous mesh800 having a plurality of tortuous paths extending through the mesh toallow nutritional formula 110 to pass through. In some embodiments, thetortuous paths may be irregular in size, shape, and/or distribution ormay be substantially regular and ordered. In some embodiments, theshapes and locations of the tortuous paths may be randomly generatedduring the manufacturing of porous mesh 800. In other embodiments, thetortuous paths and the shapes of the cross-sections of the tortuouspaths may be predetermined and, for example, designed usingcomputer-aided design packages. For example, the dimensions and theconfiguration of the tortuous paths of mesh 800 may be first modeled ordesigned using a computer-aided design (CAD) package and manufactured byusing additive manufacturing technologies, such as 3D printing. Suchmethods of making may also be used for the channels of mesh 800 in FIG.16A.

As shown in FIG. 16B, tortuous paths of porous mesh 800 may causenutritional formula 110 to pass through inlet filter 250 whiledistributing out along porous mesh 800. In this manner, rather thanpassing only through certain portions of inlet filter 250, e.g., themiddle, causing fluid channeling and/or shunting, nutritional formula110 may be more evenly distributed across outtake surface 820 of mesh800. Such distribution of nutritional formula 110 may allow nutritionalformula 110 to flow through more or substantially all of a cross-sectionof chamber 222, and thus across a broader cross-section of particles300, thus reducing the formation of channeling and/or shunting ofnutritional formula 110 through particles 300 in chamber 222.Accordingly, nutritional formula 110 would be exposed to more particles300 and to more lipase 710, potentially increasing the efficiency ofdevice 200 for hydrolyzing fats.

Filters, including tortuous path filters, mesh filters, and depthfilters, e.g., may also affect the hydrolysis of fat by breaking up thefat particles in nutritional formulas 110. Packaged nutritional formulasand pasteurized, homogenized human milk have emulsified fatpresentations so that the oily and aqueous phases do not separate duringroom-temperature storage. The emulsified fat particles may vary in size,or may coat the surface of particles 300, which may affect the abilityof the immobilized lipase on particles 300 to gain access to thetriglyceride backbone for effective hydrolysis of the triglycerides intomonoglycerides and free fatty acids. The inclusion of filters maypromote hydrolysis by breaking up the particles into smaller, moreuniform sizes.

Tortuous filters or depth filters may modify the size of the emulsifiedfat particles. By varying the filter pore size, filter type, and/orfilter depth, the emulsions may be disrupted into smaller particles. Inone preliminary study, a first sample of pasteurized, homogenized humanmilk was passed through a single-layer mesh filter, and a second sampleof pasteurized, homogenized human milk was passed through a depthfilter. In the initial experiment, passing the milk formula through themesh filter resulted in disruption of the emulsion into smallerparticles or smaller fat globules compared to the milk formula passedthrough the depth filter. In theory, it may be easier for lipase withindevice 200 to interact with smaller emulsion particles to hydrolyzefats.

Filters may also act to disrupt proteins or phospholipids surroundingthe fats within nutritional formulas. For example, as the nutritionalformula passes through a filter, the filter may break up a layercontaining phospholipids and proteins that surrounds the fats to allowthe lipase within chamber 222 to gain access to the fats more easily. Insome embodiments, one or more filters may also be coated with a proteaseto promote the break-up of proteins.

The sizes and/or diameters of the pores, channels, and/or paths of mesh800 in inlet filter 250 and/or outlet filter 260 are smaller than thediameters of particles 300, preventing particles 300 from passingthrough inlet filter 250 and/or outlet filter 260. For example, themedian or mean diameter of the pores, channels, and/or paths of porousmesh 800 may be smaller than the smallest diameter of particles 300, forexample, by about 10% to about 20%, by about 20% to about 30%, by about30% to about 40%, by about 40% to about 50%, by about 50% to about 60%,by about 20% to about 60%, by about 30% to about 60%, by about 40% toabout 60%, by about 50% to about 60%, by about 10% to about 30%, byabout 10% to about 40%, by about 10% to about 50%, or by about 10% toabout 60%, to prevent particles 300 from passing through and/or cloggingthe pores, channels, and/or paths of mesh 800. In some embodiments, thediameters or perimeters of the pores, channels, and/or paths in mesh 800may range from about 10 μm to about 100 μm, from about 10 μm to about150 μm, from about 10 μm to about 200 μm, from about 10 μm to about 300μm, from about 10 μm to about 400 μm, from about 10 μm to about 500 μm,from about 50 μm to about 300 μm, from about 50 μm to about 400 μm, fromabout 50 μm to about 500 μm, from about 100 μm to about 200 μm, fromabout 100 μm to about 300 μm, from about 100 μm to about 400 μm, or fromabout 100 μm to about 500 μm.

In some embodiments, the sizes or diameters of the pores, channels,and/or paths in mesh 800 may depend on the distribution of the diametersof particles 300. As discussed above, in some embodiments, particles 300may be sieved to filter out particles having diameters smaller than alower threshold, such as the median or mean diameter of the pores,channels, and/or paths of mesh 800. Such sieving or filtering may reducethe probability of particles 300 having diameters at the smaller end ofthe distribution that could pass through and/or clog inlet filter 250and/or outlet filter 260.

In one embodiment, both inlet filter 250 and outlet filter 260 mayinclude a traditional mesh 800, like that shown in FIG. 16A. In anotherembodiment, both inlet filter 250 and outlet filter 260 may comprise aporous mesh 800 including tortuous paths, like that shown in FIG. 16B.In another embodiment, inlet filter 250 may comprise a traditional mesh800 and outlet filter 260 may comprise a porous mesh 800. In anotherembodiment, inlet filter 250 may comprise a porous mesh 800 and outletfilter 260 may comprise a traditional mesh 800. In another embodiment,inlet filter 250 may comprise both a traditional mesh 800 and a porousmesh 800 and outlet filter 260 may comprise a traditional mesh 80 or aporous mesh 800. In another embodiment, inlet filter 250 may comprise atraditional mesh 800 or a porous mesh 800 and outlet filter 260 maycomprise a traditional mesh 800 and a porous mesh 800. In anotherembodiment, both inlet filter 250 and outlet filter 260 may comprise atraditional mesh 800 and a porous mesh 800.

In some embodiments, the thickness of inlet filter 250 and/or outletfilter 260 may range from about 0.1 mm to about 1 mm, from about 0.1 mmto about 2 mm, from about 2 mm to about 4 mm, from about 4 mm to about 6mm, from about 6 mm to about 8 mm, from about 8 mm to about 10 mm, fromabout 0.1 mm to about 4 mm, from about 0.1 mm to about 6 mm, from about0.1 mm to about 8 mm, from about 0.1 mm to about 10 mm. The thicknessmay or may not affect the flow rate of nutritional formula 110 throughdevice 200 and/or the distribution of nutritional formula 110 acrossparticles 300.

In some embodiments, mesh 800 of inlet filter 250 and/or outlet filter260 may be made of a biocompatible, inert, and/or medical polymericmaterial, for example, polyethylene. In some embodiments, inlet filter250 and/or outlet filter 260 may be a membrane filter. In someembodiments, device 200 may only have outlet filter 260 and may not haveinlet filter 250. In some embodiments, device 200 may have more than oneoutlet filter 260 and/or inlet filter 250. The diameters or perimetersof the channels or tortuous paths in mesh 800 of outlet filter 260and/or inlet filter 250 may or may not be different from each other.

In some embodiments, inlet filter 250 and/or outlet filter 260 may becoated with at least one emulsifier configured to emulsify nutritionalformula 110 as it passes through. Since nutritional formula 110 iscomposed of a complex mixture that may include, for example, proteins,carbohydrates, fat, water, minerals, and/or vitamins, and may includeliquid foods that are specially formulated and processed, the emulsifiermay emulsify nutritional formula 110 into an oil-in-liquid emulsion,with fat in nutritional formula 110 in the dispersed phase and liquid asthe dispersion medium. For example, fat droplets may be distributed inthe liquid medium by the emulsifier. Creating an emulsion of nutritionalformula 110 may facilitate the interaction between the fat molecules innutritional formula 110 and lipase 710 attached to particles 300 inchamber 222. For example, fat droplets may be attracted to a hydrophobicsurface of particles 310. The surface of particles 310 may comprise alayer of PEG coating 315 and may be wetted in the liquid medium of theemulsion, and thus lipase 710 may hydrolyze the fat molecules in theemulsion that are attracted to the surface of particle 310. In someembodiments, the type of emulsifier may depend on the composition ofnutritional formula 110. In some embodiments, multiple types ofemulsifiers may be used to coat the surface of inlet filter 250 and/oroutlet filter 260. In some embodiments, nutritional formula 110 may bepre-emulsified or may already be an emulsion before being flowed throughdevice 200. In some embodiments, an internal portion of inlet 212 may becoated with an emulsifier instead of, or in addition to, inlet filter250.

Alternative suitable emulsifiers may include, for example, proteins,hydrolyzed proteins, lecithin, phospholipids, or polyvinylpyrrylidone,or any suitable combination thereof. Lecithins used as the emulsifiermay be mixtures of phospholipids, such as phosphatidyl choline andphosphatidylethanolamine, and may be extracted from sources such as eggyolk and soybeans. Alternative emulsifiers may include diacetyl tartaricacid esters, sodium or calcium stearoyl-2-lactylate, ammoniumphosphatide, alginic acid, sodium alginate, potassium alginate, ammoniumalginate, calcium alginate, propane-1,2-diol alginate, agar,carrageenan, processed eucheuma seaweed, locust bean gum, carob gum,guar gum, tragacanth, acacia gum; gum arabic, xanthan gum, karaya gum,tara gum, gellan gum, konjac, soybean hemicellulose, cassia gum,polyoxyethylene sorbitan monolaurate, polysorbate 20, polyoxyethylenesorbitan mono-oleate, polysorbate 80, polyoxyethylene sorbitanmonopalmitate, polysorbate 40, polyoxyethylene sorbitan monostearate,polysorbate 60, polyoxyethylene sorbitan tristearate, polysorbate 65,pectins, ammonium phosphatides, sucrose acetate isobutyrate, glycerolesters of wood rosins, cellulose, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl methylcellulose, carboxy methyl cellulose, crosslinked sodium carboxy methylcellulose, enzymatically hydrolyzed carboxy methyl cellulose, sodium,potassium, magnesium, and calcium salts of fatty acids, mono- anddiglycerides of fatty acids, acetic acid esters of mono- anddiglycerides of fatty acids, lactic acid esters of mono- anddiglycerides of fatty acids, citric acid esters of mono- anddiglycerides of fatty acids, tartaric acid esters of mono- anddiglycerides of fatty acids, mono- and diacetyltartaric acid esters ofmono- and diglycerides of fatty acids, mixed acetic and tartaric acidesters of mono- and diglycerides of fatty acids, sucrose esters of fattyacids, sucroglycerides, polyglycerol esters of fatty acids, polyglycerolpolyricinoleate, propane-1,2-diol esters of fatty acids, thermallyoxidized soya bean oil interacted with mono- and diglycerides of fattyacids, sodium stearoyl-2-lactylate, calcium stearoyl-2-lactylate,stearyl tartrate, sorbitan monostearate, sorbitan tristearate, sorbitanmonolaurate, sorbitan monooleate, sorbitan monopalmitate, or invertase,for example.

As described above, nutritional formula 110 may be directed throughdevice 200 by pump 120, by gravity feeding, or via use of a syringe. Insome embodiments, nutritional formula 110 may be directed through device200 at a flow rate ranging from about 0.02 mL/min to about 2 mL/min,from about 0.4 mL/min to about 2 mL/min, from about 0.4 mL/min to about4 mL/min, from about 0.4 mL/min to about 6 mL/min, from about 0.4 mL/minto about 8 mL/min, from about 0.4 mL/min to about 10 mL/min, from about0.4 mL/min to about 12 mL/min, from about 0.4 mL/min to about 14 mL/min,from about 2 mL/min to about 6 mL/min, from about 2 mL/min to about 8mL/min, from about 2 mL/min to about 10 mL/min, from about 2 mL/min toabout 12 mL/min, from about 2 mL/min to about 14 mL/min, from about 0.02mL/min to about 4 mL/min, from about 0.02 mL/min to about 6 mL/min, fromabout 0.02 mL/min to about 8 mL/min, from about 0.02 mL/min to about 10mL/min, from about 0.02 mL/min to about 12 mL/min, from about 0.02mL/min to about 14 mL/min, from about 0.4 mL/min to about 14 mL/min, orfrom about 0.4 mL/min to about 12 mL/min.

In some embodiments, the volume of nutritional formula 110 flowedthrough device 200 may depend on the need of the subject receivingnutritional formula 110. In some embodiments, the volume of nutritionalformula 110 may range from about 1 mL to about 10 mL, from about 10 mLto about 100 mL, from about 100 mL to 250 mL, from about 250 mL to about500 mL, from about 500 mL to about 750 mL, from about 750 mL to about 1L, from about 1 L to about 2 L, from about 1 L to about 3 L, from about2 L to about 3 L, from about 1 mL to about 100 mL, from about 1 mL toabout 500 mL, from about 1 mL to about 1 L, from about 100 mL to 500 mL,from about 100 mL to 750 mL, from about 100 mL to 1 L, from about 500 mLto about 1 L, from about 500 mL to about 2 L, from about 750 mL to about2 L, or from about 750 mL to about 3 L. In some embodiments, device 200may be selected to have a volume of chamber 222 to be suitable todeliver nutritional formula 110 of a predetermined volume or at apredetermined flow rate. For example, a device 200 having a highervolume of chamber 222 may be selected to deliver a larger amount ofnutritional formula 110 or an amount of nutritional formula 110 at ahigher flow rate to an adult than that selected to deliver a smalleramount of nutritional formula 110 or an amount of nutritional formula110 at a lower flow rate to an infant.

In some embodiments, the time needed to deliver the total amount ofnutritional formula 110 through device 200, i.e., a feeding time ofnutrient formula 110, may depend on the flow rate, the volume of chamber222, and/or the total volume of nutritional formula 110 to be deliveredto the subject. For example, a faster flow rate and/or a larger volumeof chamber 222 may allow a predetermined volume of nutritional formula110 to flow through device 200 for a shorter feeding time.

In some embodiments, the feeding time may depend on the need or enteralfeeding practice suitable to the subject. In some embodiments, thefeeding time may range, for example, from about a few seconds to a fewminutes, from about a few minutes to about 30 minutes, from about 30minutes to about an hour, from about an hour to about 4 hours, fromabout 4 hours to about 10 hours, or from about 10 hours to about 12hours. In some embodiments, a shorter feeding time may be preferable forsubjects in need of nutritional formula 110.

In some embodiments, during a feeding period, nutritional formula 110flowed through device 200 may be exposed to a substantially consistentamount of particles 300 over time and may be able to react with asubstantially consistent amount of lipase 710 on particles 300. It ishypothesized that the amount of exposure of nutritional formula 110 tolipase 710 on particles 300 may be correlated to the opportunity forlipase 710 to interact with the fat molecules in nutritional formula110, which may be increased as the surface area of particles 300increase. A greater opportunity for lipase 710 to interact with the fatmolecules in nutritional formula 110 may be correlated with a higherhydrolysis efficiency of device 200. Accordingly, increased exposure ofnutritional formula 110 to lipase 710 on particles 300 may result inmore fat out of the total amount of fat in nutritional formula 110 to behydrolyzed by device 200.

In some embodiments, a residence time of nutritional formula 110 inchamber 222, i.e., the time that nutritional formula 110 is withinchamber 222 and exposed to particles 300, may affect the exposure to orinteraction between the fat molecules in nutritional formula 110 andlipase 710 on particles 300. For example, longer residence time mayallow the fat molecules to have more dwell time to move around withparticles 300 in chamber 222 or to have increased probability tointeract with and be hydrolyzed by lipase 710 on particles 300. The flowrate of nutritional formula 110 may affect the residence time ofnutritional formula 110 in chamber 222 and may affect the amount of timefor the fat molecules in nutritional formula 110 to interact with lipase710 on particles 300. A faster flow rate may drive nutritional formula110 through chamber 222 in a shorter amount of time than a slower flowrate.

In some embodiments, the amount of residence time needed may vary basedon the composition of nutritional formula 110 or the type of fat innutritional formula 110. For example, longer residence time may beneeded for a nutritional formula 110 having a higher density of fat or ahigher viscosity. In some embodiments, recycling loops may be added tothe flow of nutritional formula 110 through device 200 or through thetubes of system 100 to increase the overall residence time ofnutritional formula 110 in chamber 222. In some embodiments, increasingthe diameter of chamber 222 while maintaining the diameter of inlet 212and/or outlet 272 may increase the residence time of nutritional formula110 in chamber 222. In some embodiments, the thickness of inlet filter250 and/or outlet filter 260, and/or the diameters and/or perimeters ofthe channels and/or tortuous paths of the filters may affect theresidence time. For example, greater thickness and/or smaller diametersof the tortuous paths of outlet filter 260 may increase the residencetime. In some embodiments, longer residence time of nutritional formula110 in chamber 222 may allow more exposure to or interaction between thefat molecules and lipase 710, and thus may improve the hydrolysisefficiency of device 200, but the residence time may not be so long sothat the free fatty acids generated in nutritional formula 110 spoil.

In some embodiments, increasing the flow rate of nutritional formula 110may clear chamber 222 of nutritional formula 110 containing hydrolyzedfats and allow new nutritional formula 110 containing unhydrolyzed fatto enter chamber 222. This may free up lipase 710 to react with the newnutritional formula 110, increasing hydrolysis efficiency of device 200.However, as discussed above, increasing the flow rate of nutritionalformula 110 through device 200 may decrease the residence time ofnutritional formula 110 in device 200 and may reduce the hydrolysisefficiency of device 200. On the other hand, decreasing the flow rate ofnutritional formula 110 may increase a residence time of free fattyacids already hydrolyzed by lipase 710 in device 200, which may increasethe probability of oxidative degradation of the pre-hydrolyzed freefatty acids before ingestion. Thus, the flow rate of nutritional formula110 through device 200 may need to be designed to balance the hydrolysisefficiency of device 200, the total feeding time, and the prevention ofoxidative degradation of pre-hydrolyzed free fatty acids andmonoglycerides, and may need to be individually determined to besuitable for feeding a particular nutritional formula 110 to a subjectfor a particular feeding regimen.

In some embodiments, higher hydrolysis efficiencies may be achieved evenwhen used with faster flow rates. A higher hydrolysis efficiency and/ora faster flow rate would allow device 200 to deliver a volume ofnutritional formula 110 in a shorter amount of feeding time. This may bepreferable for patients in need of a large volume of nutritional formula110 in one or more feeding runs. By achieving higher hydrolysisefficiencies even at faster flow rates, device 200 may be able toefficiently deliver hydrolyzed triglycerides having LCTs, such asLC-PUFAs, to the subject at the time of feeding for point-of-care use,reducing the problem of oxidative degradation of free fatty acids innutritional formula 110.

In some embodiments, increasing mixing or agitation of nutritionalformula 110 in chamber 222 may increase the exposure to, or interactionbetween, the fat molecules in nutritional formula 110 and lipase 710 onparticles 300. For example, particles 300 may move under the influenceof the flow dynamics of nutritional formula 110 in chamber 222. In someembodiments, nutritional formula 110 and/or particles 300 may follow alaminar flow, a convective flow, a turbulent flow, an agitated flow, ora combination thereof in chamber 222. The type of flow achieved may alsoin part be affected by the density and/or viscosity of nutritionalformula 110 flowed through device 200. Increasing the mobility andmovement of particles 300 may increase the exposure to or interactionbetween the fat molecules and lipase 710 on particles 300. In someembodiments, headspace 223 may allow room for particles 300 to move andmix with nutritional formula 110. In some embodiments, headspace 223 mayfacilitate the mixing or may increase the turbulence or agitation ofnutritional formula 110 in chamber 222. In some embodiments, adjustingthe ratio between the shape or volume of particles 300 and the shape orvolume of chamber 222 and/or the volume of headspace 223 may increasethe mixing and movement of particles 300, and thus increase the exposureto or interaction between the fat molecules and lipase 710 on particles300. In some embodiments, device 200 may be agitated during the flow ofnutritional formula 110 manually or automatically by a shaking,twisting, tilting, or movement of device 200.

In some embodiments, increasing distribution of nutritional formula 110in chamber 222 may increase the exposure to or interaction between thefat molecules and lipase 710 on particles 300. For example, as discussedabove, the tortuous paths of porous mesh 800, shown in FIG. 16B, ofinlet filter 250 may result in a dispersed or a more even distributionof nutritional formula 110 across outtake surface 820 of porous mesh800. Such distribution of nutritional formula 110 may allow nutritionalformula 110 to flow through more or substantially all of a cross-sectionof chamber 222, and thus more or substantially all of particles 300, andmay reduce channeling or shunting of nutritional formula 110 throughparticles 300 in chamber 222, which could otherwise limit exposure. Insome embodiments, headspace 223 may also facilitate a reduction inchanneling and/or the dispersion of nutritional formula 110 by allowingparticles 300 to move, flow, and/or mix, as discussed above.

In some embodiments, pump 120 may be a peristaltic pump that drivesnutritional formula 110 under a peristaltic or inconsistent flow, whichmay increase the movement and/or mixing of particles 300 in chamber 222,and thus may increase the exposure to or interaction between the fatmolecules and lipase 710 on particles 300. Example 2, described below,shows an exemplary distribution of flow of nutritional formula 110through device 200.

Example 2: Distributed Flow of Nutritional Formula 110 Through ExemplaryDevice 200

FIG. 17 shows an experiment testing the flow of nutritional formula 110through an exemplary device 200. Exemplary device 200 used in thisexperiment was substantially similar to the devices used in Example 1. Adigital peristaltic pump 120 was used in this experiment to direct adiscontinuous flow of formula sample through exemplary particles 300 indevice 200. The formula sample was dyed with a food coloring agent sothat the flow of the formula sample could be observed. The left panel,the middle panel, and the right panel of FIG. 17 show the locations of afront of the flow profile of the formula sample at 20 seconds, 45seconds, and 75 seconds after pump 120 began pumping. As shown in FIG.17, as the formula sample entered device 200, the front of the flowprofile of the formula sample moved substantially evenly acrossparticles 300 in chamber 222. The flow of the formula sample in thisexperiment was peristaltic and when the pump was not pumping, the frontof the flow profile of the formula sample remained substantially inposition and did not continue to diffuse throughout particles 300 inchamber 222. When the pump began pumping again, the front of the flowprofile of the formula sample continued moving through particles 300 inchamber 222. This discontinuous flow was repeatedly observed during theexperiment until the entire chamber 222 of device 200 was filled withthe formula sample. The formula sample then began to exit device 200 viaoutlet 270. The total amount of time used to fill device 200 with theformula sample, as visualized, and determined at the time when theformula sample exit outlet 270 of device 200, was about 1.25 minutes.The flow rate of the formula sample was set at 2 mL/min by setting pump120, which suggests that it took about 2.5 mL of the formula sample tofill chamber 222 of device 200. No evidence of channeling was observedin this experiment.

This experiment demonstrates that the flow of the formula sample throughparticles 300 in chamber 222 is distributed approximately evenly acrossa cross-section of particles 300 in this embodiment of device 200. Asdiscussed above, such even distribution of nutritional formula 110 indevice 200 may increase the exposure to and/or interaction betweenlipase 710 attached to particles 300 and the fat molecules innutritional formula 110 and thus may improve the hydrolysis efficiencyof device 200.

In some embodiments, adjusting the mass density of particles 300 mayaffect the exposure to or interaction between the fat molecules innutritional formula 110 and lipase 710 on particles 300. For example, ifdevice 200 is placed in a vertical position, particles 300 having asmaller mass density than nutritional formula 110 may float or movetowards inlet filter 250. In such situations, the flow of nutritionalformula 110 from inlet filter 250 to outlet filter 260 may agitateparticles 300 and/or may facilitate mixing of particles 300 with theflow of nutritional formula 110. In some embodiments, having a massdensity of particles 300 that substantially matches that of nutritionalformula 110 may allow particles 300 to be dispersed or suspended innutritional formula 110, and may allow particles 300 to move with theflow dynamics of nutritional formula 110. In some embodiments, a mixtureof particles 300 having different densities may be selected so that whennutritional formula 110 is flowed through chamber 222, some particles300 may move around in a top part of chamber 222, some particles 300 maysuspend and may move around in a middle part of chamber 222, and someparticles 300 may move around in a bottom part of chamber 222, which mayincrease the mixing of particles 300 with nutritional formula 110 andmay increase the exposure to or interaction between the fat molecules innutritional formula 110 and lipase 710 on particles 300. In someembodiments, the mass density of particles 300 may not substantiallyaffect the mixing of particles 300 with nutritional formula 110.

In some embodiments, device 200 may be used in a vertical position. Ingravity feeding embodiments, device 200 may be orientated in a verticalposition for nutritional formula 110 to flow through device 200, as isshown in FIG. 17. In other embodiments, device 200 may be used in ahorizontal position. In some embodiments, device 200 may be used in avertical position with outlet 282 facing upward, or with outlet 282facing downward, or in a horizontal position.

Example 3: Comparison of Flow Rate of Exemplary Nutritional Formula 110Through Exemplary Device 200 in Different Orientations

In this example, an experiment was performed to test and compare theflow rates of nutritional formula 110 through an exemplary device 200and the hydrolysis efficiencies of device 200 when device 200 was usedin different orientations: a first vertical position with outlet 282facing upward, a second vertical position with outlet 282 facingdownward, and a horizontal position. Exemplary device 200 used in thisexperiment was substantially similar to those used in Example 1, usinginlets made of elastomer and outlets made of polycarbonate.Additionally, an O-ring gasket was used with second connector 270 ofdevice 200 so that second connector 270 could be removably fitted tobody 210 of device 200, making device 200 refillable. A total of 6formula samples, consisting of two types of commercially availablenutritional formula 110, Peptamen® and Peptamen AF®, were used for thisexperiment. Each formula sample was flowed through device 200 in therespective positions driven by pump 120 at a set flow rate of 120 mL/hr.Table 3 shows the average flow rate measured during the flow of eachformula sample. Table 4 shows the amount of hydrolyzed free fatty acidsdelivered in each formula sample.

TABLE 3 Flow rate of nutritional formula 110 with exemplary device 200positioned in three orientations Flow rate mL/hour Peptamen ® PeptamenAF ® Outlet up 125 124 Outlet down 126 125 Horizontal 125 125 Average125 125 Standard Deviation (SD) 0.4 0.3 % CV 0.3 0.3

As shown in Table 3, the average flow rates of the formula samplesflowed through device 200 at the three different orientations did notvary more than a CV of 0.3%. As used herein, CV refers to the standarddeviation divided by the mean value. Thus, a small CV indicates that theflow rate of the formula samples through device 200 was notsubstantially affected by the orientation of device 200.

TABLE 4 Amount of free fatty acids produced by exemplary device 200 atdifferent positions grams of Free Fatty Acids (FFA) delivered perserving Peptamen ® Peptamen AF ® Outlet up 6.6 g 6.6 g Outlet down 6.2 g7.1 g Horizontal 6.0 g 6.7 g Average 6.3 g 6.8 g SD 0.3 0.3 % CV 5.3 4.2

Further, as shown in Table 4, the amount of free fatty acid in theformula samples hydrolyzed and delivered by device 200 did not vary morethan a CV of approximately 5%, and thus the hydrolysis of the fats inthe formula samples was not substantially affected by the orientation ofdevice 200. The results in Tables 2 and 3 demonstrate the ability ofdevice 200 to operate in different orientations, including vertical andhorizontal.

In some embodiments, hydrolysis efficiency of device 200 may varydepending on the composition of nutritional formula 110 and thespecificity of lipase 710 to the fat in a particular nutritional formula110. In some embodiments, the hydrolysis efficiency may increase as thetemperature of nutritional formula 110 increases. For example,increasing the temperature of nutritional formula 110 from about 4° C.to about 20° C., from about 4° C. to about 25° C., from about 4° C. toabout 37° C., from about 25° C. to about 37° C., or from about 20° C. toabout 37° C. may increase the enzymatic activity of lipase 710 and mayfurther increase the thermal dynamic movement of particles 300 and/orfat molecules of nutritional formula 110 in chamber 222, which mayincrease the exposure to and/or interaction between lipase 710 and fatmolecules of nutritional formula 110.

In some embodiments, the hydrolysis efficiency of device 200 may or maynot be affected by the type of nutritional formula 110 or the hydraulicresistance of device 200 to the flow of nutritional formula 110. In oneembodiment, device 200 may be designed to provide a similar hydrolysisefficiency across a range of different commercially availablenutritional formulas 110. In some embodiments, the hydrolysis efficiencyof device 200 for commercially available nutritional formulas 110 mayrange from about 50% to about 60%, from about 60% to about 70%, fromabout 70% to about 80%, from about 70% to about 90%, from about 70% toabout 100%, from about 80% to about 90%, from about 80% to about 100%,from about 90% to about 95%, from about 90% to about 99%, from about 90%to about 100%, or from about 95% to about 100%.

Hydrolysis efficiency has also been tested on pasteurized human milk.Pasteurized human milk may contain up to 20-30% free fatty acids due tohydrolysis of the milk during storage and handling of the milk prior topasteurization. Usually no further hydrolysis occurs afterpasteurization, because the lipase that exists in human milk (bile saltstimulated lipase) is generally inactivated during pasteurization.Device 200 was tested with 30 mL of pasteurized human milk (which is atypical feeding volume used in the neonatal intensive care unit)delivered over 30 minutes (which is the standard feeding duration forpreterm infants) to measure the extent of triglyceride hydrolysis. In apreliminary experiment, device 200 was able to increase the free fattyacid content in the pasteurized human milk by approximately 25% orgreater.

In some embodiments, device 200 may introduce hydraulic resistance tothe flow of nutritional formula 110 as nutritional formula 110 flowsthrough device 200. The magnitude of hydraulic resistance to the flow ofnutritional formula 110 may be affected by a number of variables ofdevice 200, including the diameters or shapes of inlet 212 and/or outlet282; the material, thicknesses, and/or the sizes of the pores, channels,and/or paths of inlet filter 250 and/or outlet filter 260; the number,mass density, swelling, wetting characteristics, and diameters ofparticles 300; the mixing of particles 300; the volume of headspace 223;and the shape or size of chamber 222. Changing one variable of device200 or nutritional formula 110 may affect the hydraulic resistance tothe flow of nutritional formula 110, and thus may affect the flow rateof nutritional formula 110 through device 200, and may eventually affectthe hydrolysis efficiency of device 200.

Accordingly, to achieve a desired hydrolysis efficiency of device 200, anumber of different variables of device 200 may need to be designed andmanipulated. For example, in some embodiments, increasing the volume ofheadspace 223 may reduce the hydraulic resistance to or may maintain thehydraulic resistance to the flow of nutritional formula 110 at a lowermagnitude as nutritional formula 110 flows through chamber 222. Inanother example, headspace 223 may facilitate the flow of nutritionalformula 110 through particles 300 by allowing particles 300 to move orby increasing the mobility of particles 300. In another example, asnutritional formula 110 flows through particles 300, particles 300 mayswell. Headspace 223 may limit or prevent swelled particles 300 fromobstructing the pores and/or paths of inlet filter 250, and thus reducethe hydraulic resistance to or maintain a low hydraulic resistance tothe flow of nutritional formula 110. Thus, in the embodiments in whichparticles 300 may swell, headspace 223 may reduce the hydraulicresistance to the flow of nutritional formula 110 and may facilitate themaintenance of a steady flow rate of nutritional formula 110 throughdevice 200.

Examples 4-6, described below, evaluate the effect of materials of inletfilter 250 and/or outlet filter 260, the amount of particles 300, andthe diameter of chamber 222 on the flow rate of nutritional formula 110through exemplary devices 200.

Example 4: Evaluation of Effects of Exemplary Filter Materials on FlowRates of Nutritional Formula 110 Through Exemplary Devices 200

A series of test runs were performed to evaluate the effects ofexemplary materials of mesh 800 for inlet filter 250 and outlet filter260 on flow rates of nutritional formula 110. Adjustable columns ofvarious diameters were used to mimic exemplary devices 200 havingchambers 222 of different diameters. Different materials of inlet filter250 and outlet filter 260 were also tested, and the different filtertypes were fitted in the columns. A sample of 1 L Peptamen AF® wasflowed through each column using an exemplary pump 120 at a set flowrate of 120 mL/hr.

Porous plastic materials that were authorized for contact with food,compatible with gamma sterilization, and had an approximate porosity of105 μm were considered for inlet filter 250 and outlet filter 260. Twoporous plastic materials from Porex Corporation (Porex X-4906 PE orPorex POR-4744 Hydrophilic PE) were selected. Each of the two porousplastic materials was a customizable polyethylene (PE) sheet and had athickness of 0.125″ and had a porosity range of 90 μm to 130 μm. Anotherporous plastic material from Applied Separations was initiallyconsidered. This material was a hydrophilic PE sheet with a porosityranging from 20 μm to 70 μm and a thickness of 0.062″. In part due tothe higher rigidity of the thicker materials, only the two porousplastic materials from Porex Corporation (Porex X-4906 PE or PorexPOR-4744 Hydrophilic PE) were tested for use as inlet filter 250 andoutlet filter 260.

Each of the selected porous plastic materials was fitted into threeempty Omni Fit Adjustable Columns, one having a diameter of 6.6 mm, onehaving a diameter of 10 mm, and one having a diameter of 15 mm. Theeffect of each porous material on the flow rate of nutritional formula110, Peptamen AF®, through the different columns was evaluated. Based onthis evaluation, the mesh material was selected and the effects of thedensity of particles 300 and diameters of chamber 222 on the flow rateof nutritional formula 110 were then evaluated.

Each Porex porous PE sheet was cut into six disks having diameterssubstantially the same as the diameters of the three columns, i.e., apair of disks having a diameter of 6.6 mm, a pair of disks having adiameter of 10 mm, and a pair of disks having a diameter of 15 mm. Thethree columns were cleaned and dried, and each pair of plastic diskshaving about the same diameter of the corresponding column were insertedinto the filter seats of each column's inlet and outlet fittings. Theinlet and outlet fittings were then inserted into each column to furthermimic device 200. To assess the performance of the filter materials, noparticles were placed in the chamber created between the pairs of filterdisks. Each column was installed onto an enteral feeding circuit in ahorizontal orientation and fluidly connected to a pump set tubing.

Each enteral feeding circuit was then manually primed up to the inlet ofthe empty adjustable column. The pump was then set to 2 mL/min, and thetimer was started. Empty 1.5 mL vials were placed under each column tocollect measurement samples for evaluating the flow rate of Peptamen AF®in the columns. The flow rate (mL/min) for each column was measuredrandomly over 100 minutes by measuring the weight of the formuladispensed from the column and into a respective vial in 30 seconds. Theweight of the filled vials at each time point was noted and the netweight of the dispensed formula in the filled vials was obtained bysubtracting the weight of the empty vials. The weight of the dispensedformula in each vial was then used to calculate a flow rate.

The filters' effect on the flow rate was assessed. Referring to theuser's manual of pump 120, the actual flow rate of pump 120 used shouldbe within about 10% that set by pump 120. For all runs, pump 120 was setat 2 mL/min or 120 mL/hr. Therefore, for each run, the actual flow rateshould have been less than about 132 mL/hr and more than about 108mL/hr. It was desired to identify a filter type that would not cause theactual flow rate of pump 120 to fall out of the 10% variation of thepump setting when not using device 200. The results of the flow ratesmeasured for each column and porous filter combination were shown inTables 4-9 below.

TABLE 5 Flow rates for an empty 6.6 mm column with Porex X-4906 filtermaterial Flow Rate Without Beads Frit: Porex X-4906 PE 0.125″ thick,90-130 um Column 6.6 mm Dia Solution: Peptamen Percent of Pump Vol TimeFlow Rate Flow Rate Initial Flow Beaker Vol (ml) (min) (ml/min) (ml/hr)Rate (%) (ml) 0 1.9 112.5 100% 32 0.2 12 11%

TABLE 6 Flow rates for an empty 6.6 mm column with Porex POR-4744 filtermaterial Flow Rate Without Beads Frit: Porex POR-4744 PE Hydrophilic0.125″ thick, 90-130 um Column 6.6 mm Dia Solution: Peptamen Percent ofPump Vol Time Flow Rate Flow Rate Initial Flow Beaker Vol (ml) (min)(ml/min) (ml/hr) Rate (%) (ml) 5 2.4 144 100% 18 2.2 132 92% 32 2 12083% 60 1.8 108 75% 216 103 1.4 84 58% 175 Average flow (ml/hr): 102;Pump Set Point (ml/hr): 120; Variance: 15%

TABLE 7 Flow rates for an empty 10 mm column with Porex X-4906 filtermaterial Flow Rate Without Beads Frit: Porex X-4906 PE 0.125″ thick,90-130 um Column 10 mm Dia Solution: Peptamen Percent of Est. BeakerPump Vol Time Flow Rate Flow Rate Initial Flow Vol (ml) (min) (ml/min)(ml/hr) Rate (%) (ml) 0 1.9 144 100% 15 1.9 132 100% 30 2.0 120 107% 452.0 108 107% 60 1.7 104 93% 75 1.5 88 79% 90 1.2 72 64% 200 100 1.0 6054% 173 Average flow (ml/hr): 104; Pump Set Point (ml/hr): 120;Variance: 13%

TABLE 8 Flow rates for an empty 10 mm column with Porex POR-4744 filtermaterial Flow Rate Without Beads Frit: Porex POR-4744 Hydrophilic PE0.125″ thick, 90-130 um Column 10 mm Dia Solution: Peptamen Percent ofEst. Beaker Pump Vol Time Flow Rate Flow Rate Initial Flow Vol (ml)(min) (ml/min) (ml/hr) Rate (%) (ml) 0 2.3 136 100% 15 2.0 120 107% 301.7 104 93% 45 1.7 104 93% 60 1.7 104 93% 75 1.7 104 93% 90 1.7 104 93%209 105 1.7 105 94% 208 Average flow (ml/hr): 119; Pump Set Point(ml/hr): 120; Variance: 1%

TABLE 9 Flow rates for an empty 15 mm column with Porex X-4906 filtermaterial Flow Rate Without Beads Frit: Porex X-4906 PE 0.125″ thick,90-130 um Column 15 mm Dia Solution: Peptamen Percent of Est. BeakerPump Vol Time Flow Rate Flow Rate Initial Flow Vol (ml) (min) (ml/min)(ml/hr) Rate (%) (ml) 0 1.9 112 100% 12 2.0 120 107% 25 2.1 128 114% 401.9 112 100% 55 1.9 112 100% 70 2.1 128 114% 85 1.3 80 71% 200 100 1.9112 100% 197 Average flow (ml/hr): 118; Pump Set Point (ml/hr): 120;Variance: 1%

TABLE 10 Flow rates for an empty 15 mm column with Porex POR-4744 filtermaterial Flow Rate Without Beads Frit: Porex POR-4744 Hydrophilic PE0.125″ thick, 90-130 um Column 15 mm Dia Solution: Peptamen Percent ofEst. Beaker Pump Vol Time Flow Rate Flow Rate Initial Flow Vol (ml)(min) (ml/min) (ml/hr) Rate (%) (ml) 0 2.0 120 100% 15 1.9 112 100% 302.1 128 114% 45 1.9 112 100% 60 2.1 128 114% 75 1.7 104 93% 179 90 1.9112 100% 176 Average flow (ml/hr): 118; Pump Set Point (ml/hr): 120;Variance: 2%

As shown in Tables 4 and 5, the flow rates for both 6.6 mm column runswent below the lower limit. The 6.6 mm column with Porex X-4906 filtermaterial experienced a flow rate below the lower limit at the 32 minutestest point and pump 120 went into alarm due to no flow. The 6.6 mmcolumn with Porex POR-4744 filter material experienced a flow rate belowthe lower limit at the 103 minutes test point and a flow rate above theupper limit at the 5 minutes test point.

As shown in Tables 6 and 7, the flow rates for both 10 mm column runswent below the lower limit. The 10 mm column with Porex X-4906 filtermaterial experienced a flow rate below the lower limit at the 60 minutestest point and a flow rate above the upper limit at the 5 minutes testpoint. The 10 mm column with Porex POR-4744 filter material experienceda flow rate below the lower limit at the 30 minutes test point.

As shown in Tables 8 and 9, the flow rates for both 15 mm column runswent below the lower limit at one test point. However, both runsrecovered and finished within the tolerance of pump 120. The 15 mmcolumn with Porex X-4906 filter material experienced a flow rate belowthe lower limit at the 85 minutes test point. The 15 mm column withPorex POR-4744 filter material experienced a flow rate below the lowerlimit at the 75 minutes test point. Neither went above the upper limit.

The results in Tables 4-9 show that the flow rate appeared to improve asthe column diameter increased. The 6.6 mm columns experienced failuresearly in the evaluation. The components of larger diameter columns werealso found to be easier to install and handle compared to the componentsof the smaller diameter columns. The Porex POR-4744 hydrophilic PEfilter material appeared to provide more consistent flow rates than thePorex X-4906 PE filter material. The results indicate that a largerdiameter column with Porex POR-4744 hydrophilic PE filter material maybe advantageous due to more consistent flow rates of the nutritionalformula and ease of handling. The materials for inlet filter 250 andoutlet filter 260 may have properties that are consistent with the PorexPOR-4744 hydrophilic PE filter material.

Example 5: Evaluation of Effects of Exemplary Diameters of Chamber 222and Amount of Particles 300 on Flow Rates of Nutritional Formula 110Through Exemplary Devices 200

A series of test runs were performed to evaluate the effects ofdiameters of chamber 222 and amount of particles 300 on flow rates ofnutritional formula 110 in exemplary devices 200. Adjustable columnswere again used to substantially mimic exemplary devices 200 havingchambers 222 of different diameters, and the columns were filled withdifferent amounts of particles 300. Based on the evaluation of theporous filter materials in Example 4, Porex POR-4744 hydrophilic PEfilter material was used for this experiment. Additionally, due to earlyenteral feeding circuit failures for the columns having a diameter of6.6 mm, this experiment was limited to two groups of adjustable columns,one group having 3 columns with diameters of 10 mm, and the other grouphaving 3 columns with diameters of 15 mm. Pairs of disks of the selectedhydrophilic PE filter material having diameters substantially the sameas the diameters of the columns were inserted into the filter seats ofeach column's inlet and outlet fittings. Additionally, one of the threecolumns in each group was filled with 1 g, one of the three columns ineach group was filled with 2 g, and one of the three columns in eachgroup was filled with 4 g of exemplary particles 300, covalently boundwith lipase 710, between the two filter disks inside each respectivecolumn. When the columns containing particles 300 were placed in avertical orientation, the positions of the adjustable fittings of eachcolumn were adjusted so that there was approximately a length of 2 mm ofheadspace 223 above particles 300 in each column.

Each column with a particular diameter and a particle amount combinationwas installed in a horizontal orientation onto an enteral feedingcircuit and fluidly connected to a pump set tubing. One-liter samples ofPeptamen AF® of were flowed through each of the columns for about 100minutes using an exemplary pump 120 (Covidien Kangaroo EPump) set at aflow rate of 2 mL/min or 120 mL/hr. Each column was run 3 times. Duringeach fun, five measurement samples were collected at 0 minutes, 25minutes, 50 minutes, 75 minutes, and 100 minutes, and were used toobtain the flow rate of Peptamen AF® sample through each column. Theflow rates were obtained gravimetrically from the measurement samplesfor each run of each column as described in Example 4. The results ofthe obtained flow rates are shown in FIGS. 18-23.

As shown in FIG. 18, in two runs, the flow rates of the 10 mm columnsfilled with 1 g of particles 300 fell below pump 120's lower limit (108mL/hr) at the 50 minute time point. These flow rates continued downwardfor the remainder of the runs. As shown in FIGS. 19 and 20, the flowrates of the 10 mm columns filled with 2 g and 4 g of particles 300,respectively, fell below pump 120's lower limit (108 mL/hr) when testedat the 25 minutes time point. The only exception was the second run ofthe column filled with 4 g of particles 300, which fell below the limitwhen measured at the next 50 minutes time point. These flow ratescontinued downward for the remainder of the runs.

As shown in FIG. 21, the flow rates of the runs of columns with adiameter of 15 mm and filled with 1 g of particles 300 stayed withinpump 120's tolerance (i.e., 120 mL/hr+/−10%) for the duration of theruns. The only exception was the 0 minutes test point for the first run,which then evened out by the next time point and remained within thethreshold. As shown in FIG. 22, the flow rates of the 15 mm columnfilled with 2 g of particles 300 generally fell within the tolerancerange, but in run 2, the flow rate increased above pump 120's upperlimit (132 mL/hr) at the 50 minutes test point and fell below the lowerlimit at the 100 minutes test point. All other data points were withintolerance of pump 120 during the runs. As shown in FIG. 23, flow ratesof the 15 mm column filled with 4 g of particles 300 generally fellwithin the tolerance range, but in run 3, the flow rate fell below pump120's lower limit (108 mL/hr) at the 75 minutes test point and fell justbelow the lower limit at the 100 minutes test point. All other datapoints were within tolerance of pump 120 during the runs.

As shown in FIGS. 18-23, the 10 mm columns showed downward flow ratetrends, while the 15 mm columns showed more consistent flow rates. Theseresults indicate that a larger diameter of chamber 222 of device 200 waspreferable in this embodiment to maintain a stable flow rate. Thisconclusion was further supported by previous issues maintaining the flowrates in the 6.6 mm columns in Example 4. Additionally, the downwardflow rate trend for the 10 mm columns and the 15 mm columns filled with2 g or 4 g of particles 300 indicates that, for this embodiment, a lowertotal weight of particles 300 or amount of particles 300 may bepreferable to maintain a stable flow rate. This is further supported bythe more consistent flow rates of the 15 mm columns filled with 1 g ofparticles 300.

Although a chamber with a 15 mm diameter was shown as most-efficient inthis experiment, changes to particle type, size, or distribution maycause other chamber sizes to be more efficient. Additionally, changes tothe inlet and outlet may affect the optimal chamber size, as may changesto the filters or the amount of headspace provided.

Example 6: Evaluation of Effects of Exemplary Devices 200 on Flow Ratesof Nutritional Formula 110

A series of test runs were performed to evaluate the effects ofexemplary devices 200 on the flow rates of nutritional formula 110 bycomparing the flow rates of enteral feeding circuits without device 200,with an empty device 200 that did not include particles 300, and withdevice 200 containing particles 300. Exemplary device 200 used in thisexperiment was substantially similar to the devices used in Example 1.Based on the consistent flow rate of the 15 mm columns filled with 1 gof particles 300, exemplary devices 200 were assembled frompolycarbonate tubing with an interior diameter of 15 mm and customstereolithographic (e.g., 3D printing) exemplary inlet filters 250 andoutlet filters 260, substantially similar to the selected porous filterin Example 5. Enteral feeding circuits were assembled with a device 200filled with 1 g of particles 300, with an empty device 200 withoutparticles 300, and with no device 200 (i.e., just the tubing of thefeeding circuit). One-liter samples of Peptamen AF® were flowed throughthe enteral feeding circuits using an exemplary pump 120 set at a flowrate of 0.4 mL/min (24 mL/hr) and a flow rate of 2 mL/min (120 mL/hr).The flow rates of these enteral feeding circuits were measuredgravimetrically as described in Examples 4 and 5 at 30-minute intervalsfor 4 hours or until the formula ran out or the pump was stopped. Theresults of the observed flow rates compared to the upper limit and lowerlimit of tolerance (±10% variation) of pump 120 are shown in FIGS.24-26.

FIG. 24 compares the test runs without device 200 and with device 200containing 1 g of particles 300. Pump 120 was set at a flow rate of 2mL/min (120 mL/hr). FIG. 25 shows three test runs with device 200 thatdid not contain particles 300. Pump 120 was set at a flow rate of at 2mL/min (120 mL/hr). FIG. 26 shows a test run with device 200 containing1 g of particles 300. Pump 120 was set at a flow rate of 0.4 mL/min (24mL/hr).

All test runs exceeded the targeted minimum run time of 4 hours and ranuntil the 1 L sample formula bags were emptied. No circuit failures orpump alarms were observed during any of the test runs. During thetargeted minimum run time (4 hours), device 200 containing 1 g ofparticles 300 showed consistent flow rate performance with the pump setat 2 mL/min and 0.4 mL/min. Flow rate degradation was observed after 7hours during the 2 mL/min run. No flow rate degradation was observedduring the 0.4 mL/min run. The consistent flow rates of device 200 withand without particles 300 indicate that device 200 does notsignificantly impact flow rate.

In some embodiments, when inlet filter 250 and/or outlet filter 260include tortuous paths or channels, the tortuous paths or channels maybe designed to reduce the hydraulic resistance to the flow ofnutritional formula 110 as it passes through. As discussed previously,the tortuous paths may allow nutritional formula 110 to be distributedcross chamber 222, and thus may affect the hydraulic resistance to theflow of nutritional formula 110. In some embodiments, increasing thesizes or diameters, numbers, distribution, and/or adjusting the shapesof the pores, channels, and/or paths of porous mesh 800 may furtheraffect the hydraulic resistance to the flow of nutritional formula 110.In some embodiments, using additional inlet filter 250 or outlet filter260 or not using inlet filter 250 or outlet filter 260 may affect theoverall hydraulic resistance to the flow of nutritional formula 110.Accordingly, variations in filter design may affect the flow rate ofnutritional formula 110 through device 200, and it may be possible tooffset these effects by adjusting other components of device 200.

In some embodiments, reducing the diameters of particles 300 mayincrease the overall surface area of particles 300, as discussedpreviously, but may also increase the hydraulic resistance to the flowof nutritional formula 110. For example, in a given chamber 222,particles 300 having a smaller median or mean diameter may create ahigher density of polymeric material in chamber 222, and may create morepacking of particles 300, and thus may result in a higher hydraulicresistance to the flow of nutritional formula 110. Increasing the numberof particles 300 in chamber 222 may increase the hydraulic resistance.For example, for a given volume of chamber 222, a larger number ofparticles 300 may have less space to move and less mobility and/or maycrowd at the top or bottom of chamber 222, which may lead to a greaterhydraulic resistance to the flow of nutritional formula 110 and/orclogging of inlet filter 250 and/or outlet filter 260. Thus, maximizingthe overall surface area of particles 300 may need to be balanced withthe possibility of clogging of the filters and/or packing of particles300 and subsequent effect on the hydraulic resistance to the flow ofnutritional formula 110 through particles 300. In some embodiments,inert particles may be mixed with smaller particles 300 to disrupt thepacking of particles 300.

In some embodiments, particles 300 may swell when suspended innutritional formula 110 and may pack against each other due to swelling,and thus may have reduced mobility as nutritional formula 110 flowsthrough chamber 222. In some embodiments, a skewed, varied, bi-modal,multi-modal, or narrower distribution of the diameters of particles 300may promote the packing of particles 300 upon swelling. For example,particles having smaller diameters may fill the space between particleshaving larger diameters, which may further reduce the mobility ormovement of particles 300 during the flow of nutritional formula 110. Insuch situations, channeling of nutritional formula 110 may occur. Forexample, nutritional formula 110 may follow a path of least resistanceand may flow through a channel among particles 300 that has the leastamount of packing or hydraulic resistance. In this case, only lipase 710attached to particles 300 along the channel may be substantially exposedto nutritional formula 110, reducing hydrolysis efficiency. To reducethis channeling effect and/or packing of particles 300, particles 300may be made of a polymeric material that has less of a propensity forswelling, for example, swelling of less than about 1%, about 2%, about5%, about 10%, about 15%, or about 20% of the original dry particlesize.

In some embodiments, pump 120 may be a peristaltic pump that drivesnutritional formula 110 under a peristaltic, pulsatile, or discontinuousflow, which may reduce or inhibit the packing of particles 300. Forexample, nutritional formula 110 directed into chamber 222 under aperistaltic flow may increase the movement and/or mixing of particles300 in chamber 222, and thus may reduce or eliminate packing ofparticles 300. It may also allow particles 300 to pack less by notapplying a constant force on particles 300 towards outlet filter 260 andinstead introducing breaks.

Hydraulic resistance of device 200 to nutritional formula 110 may dependon the composition, density, and/or viscosity of nutritional formula110. In some embodiments, a higher viscosity and/or mass density ofnutritional formula 110 may lead to a greater hydraulic resistance tothe flow of nutritional formula 110. For example, nutritional formula110 with a higher viscosity may have more resistance to a driving forcefrom pump 120 to nutritional formula 110 and/or may have more frictionwithin tubes 122, 124, and particles 300 as nutritional formula 110flows through system 100 and particles 300. Such resistance may or maynot substantially affect the flow rate of nutritional formula 110through device 200.

In some embodiments, the flow rate selected for pump 120 or other devicemay be adjusted by a healthcare professional based on the composition,density, and/or viscosity of nutritional formula 110 before feeding. Forexample, the flow rate of nutritional formula 110 may be reduced from atypical setting to increase the residence time of nutritional formula110 in chamber 222 to increase the exposure to and interaction betweenthe fat molecules in nutritional formula 110 with lipase 710 onparticles 300. In another example, the flow rate of nutritional formula110 may be increased from a typical setting to reduce the total amountof feeding time to a patient in need of a large volume of nutritionalformula 110. In some embodiments, the flow rate of nutritional formula110 may be set by inputting a desired flow rate into pump 120. Asdescribe above, a number of different variables of device 200 may bedesigned and manipulated. Thus, device 200 may be designed to notsubstantially affect the flow rate of nutritional formula 110 set bypump 120. In some embodiments, an initial wetting resistance may existas particles 300 become wetted when nutritional formula 110 initiallyenters chamber 222. In such situations, the flow rate of nutritionalformula 110 may be affected initially but then the effect may decreaseover time.

In exemplary embodiments, the flow rate of nutritional formula 110 maybe substantially stable and/or predictable over the feeding time ofnutritional formula 110. For example, as demonstrated in Example 6, theflow rate of nutritional formula 110 may not vary more than an allowabledeviation or tolerance (e.g., about 5%, 10%, 15%, 20%, or 30% deviationfrom a set flow rate) of pump 120 or other flow driver, such as agravity feed. Example 7, described below, further demonstrates asubstantially stable flow rate of nutritional formula 110 flowed throughan exemplary device 200.

Example 7: Stability of Flow Rate of Nutritional Formula 110 FlowedThrough Exemplary Device 200

The flow rate of nutritional formula 110 in an exemplary device 200directed by a peristaltic pump 120 was monitored over 4 hours. Exemplarydevice 200 used in this experiment was substantially similar to thosedescribed in Example 1. Pump 120 was set to deliver a formula sample ata flow rate of 120 mL/hr or 2 mL/min. As shown in FIG. 27, the flow rateof nutritional formula 110 was maintained at a substantially stablelevel between about 120 mL/hr to about 125 mL/hr over a 4-hour simulatedfeeding period. The flow rate of a control in which nutritional formula110 was flowed without passing through device 200 was also monitored asa comparison to the flow rate of nutritional formula 110 flowed throughdevice 200. As shown in FIG. 27, the flow rate of nutritional formula110 flowed through device 200 was maintained between the upper limit andlower limit of the tolerance (e.g., 10% variation) of pump 120 over the4-hour simulated feeding period. Neither pump alarm or clogging ofdevice 200 was observed. This simulated feeding period of nutritionalformula 110 shows that the flow rate of nutritional formula 110 flowedthrough device 200 can be consistently maintained within the toleranceof pump 120.

Various components of device 200, including those pertaining to body210, chamber 222, headspace 223, particles 300, inlet filter 250 and/oroutlet filter 260, lipase 710 attached to particles 300, and parametersof these components, such as sizes, shapes, densities, and otherproperties discussed above, may vary and be designed for particularapplications. For example, the size of chamber 222, the size of theinlets and/or outlets, and/or number of particles 300 may be reduced fordevices intended for use with infants. Either individual component maybe modified or the proportion of the device may be shrunk or enlarged,according to use. For example, device 200 may come in infant, youth,and/or adult sizes. In some embodiments, the components of device 200may be adjusted based on the intended length of feeding time, the amountof nutritional formula 110 intended to be delivered, the amount ofLCPUFA to be delivered, or the intended flow rate of deliveringnutritional formula 110. For example, the size of chamber 222 and/ornumber of particles 300 of device 200 for an overnight enteral feedingprocedure may be different than those for a two-hour enteral feedingprocedure. A faster flow-rate device or a total nutrition device may belarger than a slower flow-rate device or one that is intended for use tosupplement a patient's diet. In some embodiments, the size of chamber222 and/or number of particles 300 of device 200 may depend on the typeof nutritional formula 110 to be hydrolyzed and processed.

The interplay of the various components of device 200, including thosepertaining to body 210, chamber 222, headspace 223, particles 300, inletfilter 250 and/or outlet filter 260, lipase 710 attached to particles300, and parameters of these components, such as sizes, shapes,densities, and other properties discussed above, may contribute to theoverall exposure to and interaction between lipase 710 in chamber 222and fat molecules in nutritional formula 110, and thus may affect thehydrolysis efficiency and/or performance of device 200. The design ofthe components of device 200 and their parameters may be adjusted toincrease the exposure to and interaction between lipase 710 in chamber222 and the fat molecules in nutritional formula 110. In someembodiments, device 200 may be designed so that the hydrolysisefficiency or performance of device 200 may not be significantlyaffected by the type or composition of nutritional formula 110. Device200 may be configured to work across a broad spectrum of formula types.In other embodiments, the design of various components of device 200 maybe selected based on the use of one particular formula type. Example 8,described below, demonstrates an exemplary range of commercial enteralformulas capable of being hydrolyzed by an exemplary device 200.

Example 8: Landscape of Enteral Formulas Tested by an Exemplary Device200

FIG. 28 shows a number of commercially available enteral formulashydrolyzed using an exemplary device 200. Exemplary device 200 used inthis experiment was substantially similar to that used in Example 3. Asdescribed herein, commercially available nutritional formulas differ intheir protein and fat content and may be classified as elemental,semi-elemental, and polymeric. Elemental formulas, for example, maycontain individual amino acids, glucose polymers, and may have a lowerfat content offering a smaller amount of calories derived fromlong-chain triglycerides. Semi-elemental formulas, for example, maycontain peptides of varying chain length, simple sugars, glucosepolymers or starch, and fat. Polymeric formulas, for example, maycontain intact proteins, complex carbohydrates, and varying types offats. In this experiment, five commercially available polymeric formulasand eight commercially available semi-elemental formulas were testedwith device 200. The volume of each formula used was 500 mL. The contentof each formula tested is depicted in FIG. 28, which shows the ratio ofmedium-chain triglycerides to long-chain triglycerides along the x-axisand shows the fat content along the y-axis.

An exemplary system 100, as shown in FIG. 1, was used to hydrolyze fats,such as long-chain triglycerides, in these nutritional formulas duringsimulated enteral feedings. Each nutritional formula was directedthrough an exemplary device 200 at a flow rate of 120 mL/hr forapproximately 4 hours. Each nutritional formula was collected at the endof the simulated enteral feeding, and the amount of hydrolyzed freefatty acid was analyzed using a quantitative colorimetric assay (Abcam®Free Fatty Acid Quantification Kit). Each nutritional formula was testedin duplicate simulated enteral feeding runs.

FIG. 29 shows the hydrolysis efficiency of device 200 when used with thenutritional formulas tested in this experiment, grouped by formula type.The polymeric formulas include Nutren® 2.0, TwoCal HN®, Nutren® 1.0,Osmolite® 1 cal, and Impact®. The semi-elemental formulas includePeptamen® 1.5, Peptamen AF®, Peptamen®, Peptamen Prebio®, Vital® 1.5,Vital 1.2 AF™, Vital® 1.0, and Impact Peptide® 1.5. As shown in FIG. 29,device 200 hydrolyzed over 80% of the fat in all of the nutritionalformulas but one. This 80% hydrolysis is remarkable given thedifferences in formula content, the fact that lipase 710 was covalentlybound to particles 300 in device 200, and the fact that the exposuretime was relatively short compared to industrial uses of lipase,particularly in light of the reduced activity of covalently bound lipasenoted in previous publications.

In some embodiments, device 200 may not significantly affect othernon-fat nutrients in nutritional formula 110, such as, for example,proteins, amino acids, carbohydrates, and/or vitamins. For example,lipase 710 attached to particles 300 may be highly specific tohydrolyzing fats in nutritional formula 110 and may not substantiallyinteract with or affect other nutrient components in nutritional formula110. In some embodiments, lipase 710 attached to particles 300 may havea high degree of purity, such that there are minimal or no otherproteins or enzymes, e.g., proteases, mixed with lipase 710, and thusthere are no other substances present in the lipase that could interactwith or affect other nutrient components in nutritional formula 110. Insome embodiments, lipase 710 may be purified over one or more rounds ofa purification process prior to binding with particles 300, or one ormore rounds of purification after binding with particles 300, to reduceor substantially eliminate other molecules or chemicals in lipase 710.In some embodiments, lipase 710 may be purified to 5%, 25%, 75%, oressentially 100% purity before or after immobilization. In someembodiments, the polymeric material of particles 300 may be inert andmay not interact with the nutrient components in nutritional formula110. Example 9, described below, further demonstrates a comparativeanalysis of the nutrients in sample nutritional formulas (i) havingpassed through an exemplary device 200 or (ii) not having passed throughdevice 200. The data shows that this embodiment of device 200 did notsubstantially affect other nutrients in nutritional formula 110.

Example 9: Comparative Analysis of Nutritional Formulas that PassedThrough Exemplary Device 200 or Did not Pass Through Device 200

This study was designed to assess the overall nutritional content ofnutritional formula after (i) having passed through an enteral feedingcircuit with an exemplary device 200 installed in-line (test) and (ii)having passed through an enteral feeding circuit without any device 200installed in-line (control). Exemplary device 200 used in thisexperiment was substantially similar to those described in Example 3,except that outlet 270 was permanently attached to body 210 (i.e., noO-ring was used, making the device 300 in Example 9 single-use). Acomprehensive analysis of nutrients was completed for two enteralformulas, Prosure® and TwoCal HN®. The nutrients analyzed are summarizedbelow in Table 11. Prosure® represents a formula with a less fat contentthat is lower in calories, while TwoCal HN® represents a formula with ahigher fat content that is higher in calories.

All samples (control and test) for nutrient analysis were flowed at theslowest recommended flow rate (0.4 mL/min), as it was hypothesized thatthe impact of device 200 on the formula samples may be greatest when theformula is in direct contact with the device 200 for the longestduration of time.

Triplicate sampling was performed to assess variation between thenutrients of the test and the control samples and variation within eachtest and control sample. A statistical analysis of the data wasperformed using an unpaired t-test. The nutrients of the test and thecontrol samples are shown in Table 11.

The test and control data sets were evaluated for each nutrient based on% relative standard deviation (% RSD). For observed % RSD in theseexperiments, the nutritional values determined were within expectedassay precision. Test and control data sets for most of the nutrientstested were generally comparable, and any differences between the testand control data sets were accounted for or expected from thevariability of the assay performance. Any differences observed did notexceed variability that would be expected for standard test assaysapplied to such complex matrices, i.e., nutritional formulas, used inthis testing. There were no nutrient differences observed consistentlybetween test and control samples across the two tested formulas.

For the nutrient tests for which a difference (p-value of larger than0.05) may were detected (indicated by an asterisk in Table 11), (i)there was no evidence of nutrient degradation, since the measured amountof nutrient in the test sample value was higher than in the controlsample, such as, for vitamin B₆ and calcium, or (ii) the difference innutrient levels between test and control samples were small whencomparing their amounts with each other and with the formula labelclaim, such as, for vitamins A, E, and C.

Thus, nutrient analysis of formulas that have passed through the device200 under simulated use conditions in comparison with a no-devicecontrol identified no significant differences between the test andcontrol samples for the effect of the feeding system on non-fatnutrients.

TABLE 11 Nutrients analyzed for comparative analysis of nutritionalformulas that passed through exemplary device 200 or did not passthrough device 200 Nutrient Energy, kcal Calories from fat, Cal Protein,g Total fatty acids, g EPA, g DHA, g Omega-3 fatty acids, g Omega-6fatty acids, g Carbohydrates, g Dietary fiber, g Fructooligosaccharide,g L-carnitine, mg Vitamin A, IU* Vitamin D, IU Vitamin E, IU* Vitamin C,mg* Vitamin B₆, mg (Pyridoxine)* Vitamin B₁₂, mcg Folic acid, mcgPantothenic acid, mg Biotin, mcg Sodium, mg Potassium, mg Chloride, mgCalcium, mg* Phosphorus, mg Magnesium, mg Riboflavin, mg (Vitamin B₂)Ash, g Moisture (Water, mL)

Device 200 may be designed for point-of-care use. For example, device200 may be designed to be used with standard enteral feeding devices fordelivering nutritional formula 100 to a subject in need of fatty acidnutrients in a clinic or a hospital. In some embodiments, device 200 maybe used in non-clinical settings, such as at the subject's home,long-term or short-term care facility, or at a place the subject visitsregularly. The fat, including triglycerides having LC-PUFAs, innutritional formula 110 is “digested” or pre-hydrolyzed by device 200right before feeding and is delivered in a form ready for absorption inindividuals who lack pancreatic lipase or the physiological capacity todigest or absorb fat. Such delivery of pre-hydrolyzed nutritionalformula 100 using device 200 prior to ingestion may provide directdelivery of hydrolyzed and absorbable fatty acids to the GI tract of thesubject, leading to improved delivery and absorption efficiency.

The use of device 200 may also prevent the problem of oxidativedegradation of free fatty acids in a pre-hydrolyzed nutritional formula110 and thus may prevent the development of a rancid taste, odor, ortexture in the nutritional formula after hydrolysis. Specifically, it isthe hydrolysis of fats into short-chain aldehydes and ketones that areobjectionable in taste and odor.

Industrial-scale utilization of immobilized lipase for fat hydrolysisrequires a water-oil interface to release free fatty acids. The freefatty acids are then re-esterified to form triglycerides since the freefatty acids themselves are unstable for any substantial period of time.Industrial-scale immobilization tends to be time-consuming, inefficient,and requires significant operator manipulation. The use of device 200 isgenerally with complex mixtures containing, for example, proteins,carbohydrates, fat, water, minerals, and/or vitamins, which may includeliquid foods that are specially formulated and processed.

By delivering pre-hydrolyzed absorbable free fatty acids at the point ofcare, device 200 may also reduce or eliminate the need and/or risks oftaking of porcine-derived pancreatic enzyme or microbial enzyme productsduring the feeding of nutritional formula 110. Further, as discussedabove, the amount of residence time of nutritional formula 110 in device200 may be adjusted and may be balanced with the hydrolysis efficiencyof device 200 by adjusting the flow rate to reduce or prevent oxidativedegradation of pre-hydrolyzed free fatty acid in nutritional formula110. Exemplary timespans between the exposure of nutritional formula 110to lipase and ingestion of the pre-hydrolyzed formula by a patient arediscussed in International Patent Application No. PCT/US2013/026063,filed Feb. 14, 2013, and U.S. patent application Ser. No. 14/378,856,filed Aug. 14, 2014, both of which are herein incorporated by referencein their entireties.

System 100 and device 200 allow fats in nutritional formula 110 to bepre-hydrolyzed ex vivo, prior to ingestion, and to match the time ofhydrolysis of fat with enteral feeding, leading to reliable, efficient,and consistent delivery of absorbable beneficial fats to the subject.System 100 and device 200 may provide healthcare professionals anadvantageous option for feeding patients in need of additional caloriesand essential fatty acids, such as DHA and EPA.

In some embodiments, device 200 may be disposable and intended for asingle use. In other embodiments, device 200 may be reusable for anumber of feeding runs before disposal. In such embodiments, device 200and/or the tubes, e.g., first tube 122 and enteral tube 124, may becleaned before a new feeding run by flushing or purging a solutionthrough device 200 and/or the tubes. For example, pump 120 may operateon an automatic mode to flush or purge a solution through device 200and/or the tubes to adequately empty nutritional formula 110 left indevice 200 and/or the tubes from a previous feeding run. This flushingor purging would allow device 200 and/or the tubes to be used more thanonce before disposal.

In some embodiments, particles 300 may be disposable. For example, aftera feeding run of nutritional formula 110, used particles 300 may bedisposed of and device 200 may be sterilized and/or cleaned, and for anext feeding run of nutritional formula 110, new, unused particles 300may be packaged under dry conditions in chamber 222 of device 200. Insuch embodiments, the remainder of device 200 may be sterilizable.

Patients suffering from EPI (insufficient production of exocrinepancreatic enzymes) and/or gastrointestinal or liver dysfunction have areduced ability to hydrolyze and/or absorb long-chain triglycerides. Asa result, they might have maldigestion and malabsorption of lipids,which may lead to reduced caloric intake, significant weight loss,LC-PUFAs deficiencies, and/or GI symptoms, and may be deprived of thebenefits associated with ingestion of LC-PUFAs, such as DHA, EPA, AA,etc. System 100 and device 200 may be used for feeding nutritionalformula 110 having pre-hydrolyzed triglycerides of DHA, EPA, and/or AA,to patients having compromised pancreatic output. For example, system100 and device 200 may be used to increase the intake of DHA, EPA, andAA in the plasma of these patients. In some embodiments, since healthysubjects may also benefit from increased absorption of LC-PUFAs, e.g.,by reducing the risk of cardiovascular disease, system 100 and device200 may be used for feeding a healthy subject nutritional formula 110.In some embodiments, system 100 and device 200 may be used to increasethe intake of DHA, EPA, and AA in the plasma of infants, aging adults,or people with acute or chronic conditions that may impact fathydrolysis and/or absorption.

In some embodiments, system 100 and device 200 may be used to increasethe intake of hydrolyzed fatty acids for patients having one or morediseases, including for example, Alzheimer's disease (AD), bipolardisorder (BP), depression, major depressive disorder (MDD), post-partemdepression, sepsis, acute respiratory stress, wound healing, cancer,cardiovascular disease, stroke, Parkinson's disease, schizophrenia,diabetes, multiple sclerosis, and chronic inflammatory diseases, such asrheumatoid arthritis, systemic lupus erythematosus, and inflammatorybowel disease. In some embodiments, system 100 and device 200 may beused for feeding patients who cannot obtain nutrition by mouth, areunable to swallow safely, or otherwise need nutritional supplementation.In some embodiments, system 100 and device 200 may be used to reduce theneed for parenteral nutrition. The use of enteral nutrition may bepreferred when possible, as it reduces the risk of generating infection,undesirable immune response, and/or atrophy of the GI tract. In someembodiments, system 100 and device 200 may be used for feeding patientswith prematurity, failure to thrive, malnutrition, neurologic andneuromuscular disorders, inability to swallow, anatomical andpost-surgical malformations of the mouth and esophagus, cancer,digestive, and/or metabolism disorders. In some embodiments, system 100and device 200 may be used for improving and/or supporting the therapiesof other diseases, such as cancer, by providing fatty nutrient topatients.

Additional advantages and benefits of system 100 and device 200 may alsoinclude delivering pre-hydrolyzed fats at a high efficiency. Forexample, about 70% to over about 90% of fats in nutritional formula 110may be hydrolyzed after passing through device 200, as shown in FIG. 29.The hydrolysis efficiency of system 100 and device 200 may be maintainedfor very complex nutritional formulas having various nutrients. Suchhigh hydrolysis efficiency of device 200 may reduce the total volume ofnutritional formula 110 that needs to be delivered to the patient.Further, as discussed previously, nutritional formula 110 may bedelivered at a flow rate, for example, ranging from 0.4 mL/min to about8 mL/min or higher. Under such flow rates, device 200 may allow thedelivery of a typical volume, e.g., ranging from about 1 mL to about 10mL, from about 10 mL to about 100 mL, from about 100 mL to 250 mL, fromabout 250 mL to about 500 mL, from about 500 mL to about 750 mL, fromabout 750 mL to about 1 L, from about 1 L to about 2 L, from about 1 Lto about 3 L, from about 2 L to about 3 L, from about 1 mL to about 100mL, from about 1 mL to about 500 mL, from about 1 mL to about 1 L, fromabout 100 mL to 500 mL, from about 100 mL to 750 mL, from about 100 mLto 1 L, from about 500 mL to about 1 L, from about 500 mL to about 2 L,from about 750 mL to about 2 L, from about 750 mL to about 3 L, or fromabout 3 mL to about 1 L of nutritional formula 110 containingsubstantially pre-hydrolyzed fat within seconds, minutes, or hours. Suchhigh efficiency of delivering nutritional formula 110 is preferable toimprove the quality of life for patients, especially for patients inneed of large volumes of nutritional formula 110. Examples 10-12,discussed further below, demonstrate the high hydrolysis and deliveryefficiencies of system 100 and device 200 for a wide range of enteralformulas.

In some embodiments, delivering pre-hydrolyzed nutritional formula 110by using system 100 and device 200 may allow normalization of thecaloric intake and fatty acid balance and absorption of a patient, suchas the most difficult to digest and absorb LC-PUFAs, for example DHA,EPA, and AA. This may advantageously provide a more controlled optionfor healthcare providers to improve their management and treatment ofpeople with compromised pancreatic output or lipid malabsorption.Examples 13-15 demonstrating the use of system 100 and device 200 forimproving the free fatty acid intake and balance are discussed furtherbelow.

Additional Examples of System 100 and Device 200 Example 10: In VitroHydrolysis of Enteral Formula Fats Using Exemplary Device 200, ShowingSubstantially Steady Hydrolysis Efficiency

Two experiments on the hydrolysis of triglycerides in two samples ofenteral formula Peptamen AF® were performed using an exemplary device200. Exemplary devices 200 used in this experiment was substantiallysimilar to those described in Example 1. Each experiment simulated anenteral feeding over a period of time. The first experiment tested afirst sample of 250 mL of Peptamen AF® over a feeding period of 2 hours.The second experiment tested a second sample of 500 mL of Peptamen AF®over a feeding period of 4 hours. The flow rate of the enteral formulain the two experiments was maintained at 2 mL/min. Testing samples werecollected at a plurality of time points during the feeding period ofeach experiment, and the amount of fatty acid was analyzed using ultraperformance liquid chromatography-tandem mass spectrometer (UPLC MS) ateach time point.

As shown in FIG. 30, for the first experiment, the cumulative amount offree fatty acid in the sample increased almost linearly over the feedingperiod of the experiment, as shown by the approximately diagonal line,indicating a substantially steady hydrolysis efficiency. The amount offree fatty acids delivered by the end of the experiment was about 7.3 gout of the total amount of 7.7 g of free fatty acids (shown as ahorizontal line in FIG. 30) that could have possibly been generated fromthe amount of triglycerides in the nutritional formula flowed throughdevice 200. This demonstrates a hydrolysis efficiency of about 95%, asis demonstrated graphically with the diagonal line nearly intersectingthe total horizontal line by the end of the experiment. The result inFIG. 30 shows that device 200 can efficiently hydrolyze triglycerides in250 mL enteral formula for a shorter period of feeding time of about 2hours or less at a substantially steady rate.

As shown in FIG. 31, for the second experiment, the cumulative amount offree fatty acid in the sample also increased almost linearly over theperiod of the experiment, again showing a substantially steadyhydrolysis efficiency. The amount of free fatty acids delivered at theend of the experiment was about 17.5 g out of the total amount of 18.2 gfree fatty acids (shown again as a horizontal line in FIG. 31) thatcould have possibly been generated from the amount of triglycerides inthe nutritional formula flowed through device 200, rendering ahydrolysis efficiency of about 96%. The result in FIG. 31 shows thatdevice 200 can efficiently hydrolyze triglycerides in 500 mL enteralformula for a slightly longer period of feeding time of about 4 hours orless at a substantially steady hydrolysis efficiency.

Example 11: Comparison of Ex Vivo Hydrolysis Efficiency of ExemplaryDevice 200 with Porcine-Derived Pancreatic Enzyme Capsules (PERTCapsules)

Hydrolysis of fats in three samples of Peptamen AF® by an exemplarydevice 200 and PERT products was performed and compared. Exemplarydevices 200 used in this experiment were substantially similar to thosedescribed in Example 1. PERT products are a combination of variouslipase, protease, and amylase enzymes. For the first sample, device 200was used for the hydrolysis of 237 mL of Peptamen AF® for a simulatedenteral feeding of about 2 hours. A flow rate of 2 mL/min was usedthroughout the feeding. No alarm from pump 120 or clogging was observedduring the feeding for device 200.

For the second and third samples, two types of commercially availablePERT capsules were used for the hydrolysis. The second sample washydrolyzed using 4 capsules of ZenPep® (80,000 units lipase; 272,000units protease; 436,000 units amylase; Aptalis), which is an entericallycoated product. The third sample was hydrolyzed using 3 tablets ofViokace® (62,640 units lipase; 234,900 units protease; 234,900 unitsamylase; Aptalis). The PERT capsules were added directly into the secondand third sample enteral formula bags, in order to maximize exposuretime of the PERT products to the enteral formula, each of whichcontained one can of 250 mL of Peptamen AF®. In contrast to device 200,where the enteral formula passed through the device, the PERT productswere mixed into the formula bags in order to maximize exposure andpotential hydrolytic capacity of the PERT enzymes to the enteralformula.

Samples of each formula hydrolyzed using device 200 were collected at 0,30, 60, 90, and 120 minutes during the hydrolysis process, and fathydrolysis in each sample was evaluated at each time point using aquantitative colorimetric assay (Abcam® Free Fatty Acid QuantificationKit) to measure the amount of free fatty acids. FIG. 32 shows the amountof free fatty acid detected in each formula sample at each time point.As shown in FIG. 32, the cumulative amount of free fatty acid deliveredby exemplary device 200 by the end of the experiment almost equaled theamount of free fatty acids that could have possibly been generated ifall of the triglycerides in the formula sample had been hydrolyzed. Thisresult agrees with the results depicted in FIGS. 30 and 31 and showsnear-complete hydrolysis of the triglycerides available in thenutritional formula. The free fatty acid in the second formula samplegenerated using ZenPep® capsules remained at less than 1 gram (less than10% hydrolysis) over the course of the experiment. The amount of freefatty acid in the third formula sample generated using Viokace® wasundetectable using the assay and thus does not appear in FIG. 32.

FIG. 33 shows calculated hydrolysis efficiencies in the three formulasamples discussed in regards to FIG. 32. As shown in FIG. 33, in thefirst formula sample, exemplary device 200 hydrolyzed over 90% of thefat starting at the 30-minute time point. In the second formula sample,ZenPep® capsules only hydrolyzed about 10% of the fat by the end of theexperiment, reaching only a high of 29% at the 30 minutes time point. Inthe third formula sample, hydrolysis of fat by Viokace® capsules wasundetectable. The results demonstrate that device 200 has superiorefficiency in hydrolyzing fat in enteral formulas compared to PERTcapsules.

Example 12: Hydrolyzing Fat in Nutritional Formulas of Different VolumesUsing Exemplary Device 200

A series of experiments on the hydrolysis of triglycerides in enteralformula Peptamen AF® were performed using an exemplary device 200.Exemplary device 200 used in this experiment was substantially similarto that used in Example 3. Peptamen AF® formula contains an equal amountof triglycerides with MCT and triglycerides with LCT. A 500 mL PeptamenAF® solution contains a total of 27.4 g fat, including 1.2 g EPA and DHAfrom the triglycerides. One experiment simulated an enteral feeding runof 500 mL Peptamen AF® over 1 hour at a flow rate of 8 mL/min, oneexperiment simulated an enteral feeding run of 500 mL Peptamen AF® over2 hours at a flow rate of 4 mL/min, one experiment simulated an enteralfeeding run of 500 mL Peptamen AF® over 4 hours at a flow rate of 2mL/min, one experiment simulated an enteral feeding run of 250 mLPeptamen AF® over 10 hours at a flow rate of 0.4 mL/min, and oneexperiment simulated an enteral feeding of 1 L Peptamen AF® over 8 hoursat a flow rate of 2 mL/min. The flow rate of the formula samples wasmaintained throughout the simulated feedings with no alarms detected.

As shown in FIG. 34, device 200 efficiently hydrolyzed over 90% of fatin 500 mL Peptamen AF® over the course of 2 and 4 hours, over 90% of fatin 250 mL Peptamen AF® over the course of 10 hours, and about 90% of fatin 1 L Peptamen AF® over the course of 8 hours. The hydrolysis of fat in500 mL Peptamen AF® delivered over the course of 1 hour also showed highefficiency. The results show that device 200 may hydrolyze and deliver asubstantial percentage of fats in nutritional formula 110 even over ashorter 1 to 2 hour feeding under a faster flow rate, which couldpotentially reduce the need for longer, overnight enteral feedings.

Example 13: Testing the Efficacy of Lipase 710 Attached to Particles 300for Digestion of Long-Chain Polyunsaturated Fatty Acid (LC-PUFA) inYoung Pigs with Total Pancreatic Insufficiency

This experiment evaluated whether the absorption of total fats andlong-chain polyunsaturated fatty acids (LC-PUFAs) from infant formulawas enhanced when the formula was pre-hydrolyzed with Rhizopus oryzaelipase immobilized on acrylic beads (an exemplary lipase 710 covalentlyattached to exemplary particles 300, substantially similar to theparticles 300 described in Example 1), herein referred to as iRO, justbefore consumption. This experiment was performed in a porcine model ofpancreatic insufficiency (young pigs with total pancreaticinsufficiency). The porcine model was chosen since at the functional anddevelopmental level, humans and pigs share many similarities with regardto the gastrointestinal tract, genitourinary structures, and developmentof the brain and pancreas. Surgical ligation of pancreatic ducts inyoung pigs causes impaired excretion of pancreatic enzyme, includingbile salt stimulated lipase, and thus mimics conditions in pre-termand/or full term human babies or individuals with chronic malfunction ofexocrine pancreas, such as CF patients, patients after oncology surgery,or elderly subjects. As used herein, EPI pigs are used to refer to thisporcine model.

Pancreatic duct ligation was performed on 20 pigs to create exocrinepancreatic inefficiency (EPI) for this experiment. EPI typically fullydevelops three to four weeks after the surgery. Development of completepancreatic insufficiency was confirmed by arrested growth anddevelopment of steatorrhea. However, out of 20 operated pigs, only 17EPI pigs developed complete pancreatic insufficiency and were used inthis experiment. The 17 EPI pigs (male) and 6 healthy pigs (male) weremaintained on a 12-hour day-night cycle, with light from 6 AM to 6 PMand darkness from 6 PM to 6 AM.

Nutritional formula having fat pre-hydrolyzed with iRO was divided into4 daily feedings, and its efficacy was tested in young, growing EPI pigsthat would be developmentally comparable to human babies 3-6 months ofage.

As shown in FIG. 35, the 6-week treatment study was proceeded by aninitial adaptation period of two weeks. Prior to pancreatic ductligation surgery, following surgery, and prior to the initial adaptationperiod of this experiment, all pigs were fed a standard pig diet thatcontained 17.5% crude protein, 3.9% crude fibre, 3.5% crude fat, 5.2%ash, together with 5000 IE/kg vitamin A, 500 IE/kg vitamin D, 85 mg/kgvitamin E. Feeding was done twice daily (2.0% body mass per meal) from 9AM to 10 AM and from 5 PM to 6 PM.

During the adaptation period, all pigs were fed NAN Pro 1 Gold (Nestle)formula (NAN formula) enriched with long-chain polyunsaturatedtriglycerides (TG-LCPUFA): 1% docosahexaenoic acid (DHA) and 2%arachidonic acid (AA) from fish oil. Thereafter, in this experiment, theformula was enriched with 1% TG-DHA and 2% TG-AA from fish oil,resulting in a final fat content of about 31%.

A 6-week treatment period followed the initial adaptation period. Duringthe 6-week treatment period, EPI pigs were randomized into 2 groups. Inthe control group, EPI pigs were fed with enriched formula only,referred to as non-hydrolyzed drink (ND, n=6). In the treatment group,EPI pigs were fed formula pre-hydrolyzed with iRO, referred to aspre-hydrolyzed drink (PND, n=7). In a second control group, healthy pigswith intact function of the exocrine pancreas were enrolled and fed withLC-PUFA enriched formula only (ND, n=6).

To generate the PND, a mesh bag filled with iRO was placed into theenriched infant formula (ND) and mixed with an automatic stirrer for upto 15 minutes at a temperature range from about 30° C. to about 37° C.to allow substantially complete fat hydrolysis. For a single meal for anEPI pig (100 g formula powder diluted in 300 mL water), one mesh bagwith 1 g of iRO was used. When hydrolysis was finished, the mesh bagswere removed from the bucket and discarded. The size specification ofiRO ensured that the beads could not migrate outside of the mesh bag,and the mesh bag prevented any leakage of iRO to the formula. Whenpre-hydrolysis was complete, the mesh bag was removed, and the PND wasready for consumption.

The action of the iRO was intended to mimic pancreatic lipase and togenerate free fatty acids and monoglycerides, similar to those thatwould be found after the action of endogenously secreted pancreaticlipase in the small intestine. The point-of-care approach in which PNDwas generated and supplied right before ingestion also addressed thefree radical oxidation of free fatty acids from ND and preventeddevelopment of a rancid taste or odor. Thus, the benefit of thepoint-of-care approach was that fats, including triglycerides havingLC-PUFAs were “digested” or pre-hydrolyzed just before drinking and thuswere made available for absorption by the GI tract that would otherwiselack the physiological capacity to digest fat.

The effect of pre-hydrolysis of dietary fat was monitored by assessingreduction of total and polyunsaturated fatty acids (PUFA) in fecal fats,together with increases in the absorption of AA and DHA expressed aschanges from the control group in the plasma, visceral tissue (liver,fat), heart, and neuronal tissues (hippocampus) in the pigs. Presence offats in fecal matter was interpreted as an indication that the fats hadfailed to be absorbed by the pigs.

The results of this study demonstrated no mortality, adverse clinicalsigns, or pathologic macroscopic or microscopic findings along the gutor in the liver following the six-week administration of pre-hydrolyzedformula, including administration of monoglycerides and free fatty acidsinstead of triglycerides.

13.1 Testing Design and Procedures:

13.1.1 Pre-Treatment Period (7-10 Days)

Approximately 7 days before the adaptation period, 23 pigs weretransitioned from regular food to formula feeding. Through the course ofthis experiment, 4 EPI pigs were eliminated: one due to sickness, onedue to improper gavage, and two due to pancreatic double ductdevelopment. No loss was recorded in the healthy group of pigs. Thus,the total number of pigs included in final study analysis was 13 EPIpigs and 6 healthy pigs.

13.1.2 Adaptation Period (14 Days)

During this period, all EPI pigs and healthy pigs were given warm liquidND enriched with 1% triglyceride having DHA (TG-DHA) and 2% triglyceridehaving AA (TG-AA) 4 times per day. The total daily formula consumptionwas measured every day and during the entire experiment. On day 1 (1stday of the experiment) of the Adaptation period, body weights wererecorded before the morning meal. Stool and blood samples were collectedon the last two days of this period.

13.1.3 Treatment Period (6 Weeks)

During this period, EPI pigs were randomized into two groups, based onthe body weight and willingness to consume formula:

-   -   1) Control EPI group (EPI): six EPI pigs were fed with enriched        non-hydrolyzed formula.    -   2) iRO group (EPI+iRO): seven EPI pigs were fed with formula        pre-hydrolyzed with iRO.    -   3) Healthy control group (Healthy): six healthy pigs of the same        age and breed were fed enriched formula only.

On day 1 of the each week of the 6-week treatment period, pigs wereweighed before the morning meal. Three 24-hour stool collections wereperformed during the last three days of the week 1, week 4, and week 6of the treatment period. On days 7, 28, and 42 of the treatment period,pre-prandial blood samples after an overnight fast were collected.

Weights of collected 24-hour stool samples were recorded, and a smallfraction from each sample was measured for total fat and LC-PUFAs.

For measurement of LC-PUFAs, fecal, plasma, and tissue samples wereanalyzed using a gas chromatography-mass spectrometry (GC-MS) method.

Five mL blood samples were collected on the respective days beforefeeding. The samples were analyzed for LC-PUFA, triglyceride (TG),cholesterol, low-density lipoproteins (LDL), high-density lipoproteins(HDL), and non-esterified fatty acids (NEFA) content.

At the end of the experiment, the pancreatic area and the involutedpancreas of each pig was examined for pathological changes, togetherwith the gastrointestinal tract and liver, kidney, and heart.

Statistical analysis was performed on the data generated from this studyusing the ANOVA analysis of variance of the SAS program and ordinary oneway ANOVA and ANOVA paired t-test using Prism Graph program. Differenceswere considered significant if p≤0.05. All data are expressed as amean±standard deviation (±SD).

13.2 Results

13.2.1 Effect of the Consumption of PND on Stool Weight, Appearance, andTotal Fat Content

Destruction of exocrine pancreatic function in EPI pigs resulted inmaldigestion and malabsorption that caused pronounced steatorrhea andvoluminous feces with an increased number of stools. FIG. 36A shows anexemplary stool sample of EPI pigs fed with ND, and FIG. 36B shows anexemplary stool sample of EPI pigs fed with PND. As shown in FIG. 36Aand FIG. 36B, in EPI pigs fed with PND, absorption of fat was improvedbased on visible changes in stool appearance (fatty stool disappearance)and also a decrease in weight.

As shown in FIG. 37, when total fat was measured in stool dry mattersamples, the difference between EPI pigs that were consuming PND vs. NDwas more pronounced (EPI: 66.7±24.6% vs. EPI+iRO: 37.9±18.6 g/24 h;n=6-7; p<0.02; mean of three 24 h collections during the last 3 days ofthe study). There was 43% less fat in the stool samples from the EPI+iROgroup compared to the EPI group, suggesting improved absorption of fatthat resulted in approximately an additional 243 calories consumed perday. In healthy control pigs, fat content was 13.83±2.4%.

As shown in FIG. 38A and FIG. 38B, formula intake and body weight weresubstantially the same in EPI group and EPI+iRO group. As expected, theEPI pigs didn't grow, since the formula had only pre-hydrolyzed fat andnot the proteins that are necessary for growth and increased body mass.Healthy pigs with intact function of exocrine pancreas were growing 2-4kg/week.

For estimation of LC-PUFA fat content, stool was collected on days 5, 6,and 7 on the 6th week of treatment, and individual LC-PUFA content wasmeasured. A summary of the results is shown in Table 12.

TABLE 12 Summary of fecal LC-PUFA levels in pigs from EPI group fed withND or PND and healthy pigs fed with ND. EPI EPI + iRO % Change Healthyfed ND fed PND EPI vs. fed ND n g/100 g FA EPI + iRO g/100 g FA LA 0.467± 1.01 0.126 ± 0.07* ↓73 0.047 ± 0.02** ALA 0.028 ± 0.06 0.046 ± 0.08 —0.033 ± 0.02 AA 0.674 ± 0.56 0.295 ± 0.41* ↓66 0.114 ± 0.08** EPA 0.012± 0.01 0.008 ± 0.01 ↓44 0.006 ± 0.01 DHA 0.734 ± 0.19 0.364 ± 0.31* ↓500.194 ± 0.07** Σ (n-3) 2.585 ± 0.20 1.605 ± 0.34* ↓38 0.853 ± 0.16** Σ(n-6) 4.321 ± 1.20 2.024 ± 0.58* ↓53 0.961 ± 0.23**

The data shown in Table 12 is the mean±SD of LC-PUFA levels in stoolsamples collected on the last 3 days of the last, week 6 of thetreatment (n=6-7/group) (* p<0.05 EPI vs. EPI+iRO; ** p<0.05 EPI vs.Healthy). As shown in Table 12, significant reduction of 38% and 53% infecal omega-3 and omega-6 LC-PUFA was demonstrated in the EPI+iRO groupfed with pre-hydrolyzed formula when compared with the EPI group fedwith non-hydrolyzed formula. Similarly, 66% and 50% reductions in fecalAA and DHA levels, respectively, were recorded in the EPI+iRO groupcompared to the EPI group. These data indicate that the inability of EPIpigs to absorb fat was at least partially reversed by feeding withformula pre-hydrolyzed with iRO.

13.2.2 Effect of Pre-Hydrolysis on Blood Lipid Profile

An important finding of this study was that the blood lipid profile inthe treatment group fed with PND for 6 weeks was substantiallynormalized to that of healthy pigs, as shown in Table 13. This resultsuggests not only increased absorption of fat, but also propermetabolism of fat that resulted in substantially normal blood levels ofTG, cholesterol, HDL, and LDL. All EPI pigs had normal blood glucosethat was substantially the same as in healthy pigs, confirming thatendocrine pancreatic function was preserved and not affected by surgery.

TABLE 13 Triglycerides, cholesterol, HDL, and LDL plasma levelsfollowing 6 weeks of feeding of EPI pigs with ND/PND and healthy pigswith ND TG Cholesterol HDL LDL Groups mmol/L mmol/L mmol/L mmol/LHDL/LDL Healthy 0.51 ± 0.25 4.13 ± 0.68 2.04 ± 0.31 1.27 ± 0.33 1.66 ±0.33 EPI 0.22 ± 0.07 2.69 ± 0.56 1.46 ± 0.41 0.69 ± 0.35 2.63 ± 1.34(fed ND) EPI + iRO 0.45 ± 0.17* 4.13 ± 1.35* 1.92 ± 0.42* 1.12 ± 0.51*1.82 ± 0.70 (fed PND)

Data shown in Table 13 is the mean±SD, in cohorts: healthy pigs n=6, EPIn=6, EPI+iRO n=7, for TG, cholesterol, HDL, and LDL collected frompre-prandial samples after 6 weeks of feeding of with ND or PND. Healthypigs were fed with ND. The p-value is *p<0.05 for difference between EPIand EPI+iRO groups, unpaired t-test. HDL=high-density lipoproteins;LDL=low-density lipoproteins; TG=triglycerides.

13.2.3 Plasma and Tissue Changes in LC-PUFA Levels

13.2.3.1 Changes in Plasma and RCB LC-PUFA Levels

Feeding with formula containing pre-hydrolyzed fat resulted in positivechanges in plasma PUFA levels, as shown in FIG. 39 and Table 14.

TABLE 14 Plasma LC-PUFA concentration upon consumption of pre-hydrolyzedformula for 6 weeks. Sum FA LA ALA AA EPA DHA Groups g FA/100 g g FA/100g g FA/100 g g FA/100 g g FA/100 g g FA/100 g Healthy 0.27 ± 0.04*  59 ±9.8*  1.9 ± 0.5* 35.0 ± 4.0* 0.7 ± 0.1 10.5 ± 1.2* (Control) EPI 0.16 ±0.03  35.9 ± 7.7  1.0 ± 0.3 17.0 ± 4.0  0.7 ± 0.4 3.2 ± 0.7 EPI + iRO0.23 ± 0.07* 47.6 ± 14.8*  1.4 ± 0.5*  27.4 ± 12.5* 0.7 ± 0.5 4.7 ± 2.2

Data shown in Table 14 is a sum of the polyunsaturated free fatty acidconcentration (mean±SD) in healthy pigs (n=6, EPI n=6, and EPI+iRO n=6)for sum of all FA, measured in pre-prandial blood samples collectedafter 6 weeks of feeding of EPI pigs with ND or PND. Healthy pigs werefed with ND. The p-value is *p<0.05 for difference between groups, ANOVApaired t-test (p=0.091).

As shown in FIG. 39 and Table 14, the concentration of total free fattyacid in circulation was significantly higher in EPI pigs fed for 6 weekswith formula pre-hydrolyzed with iRO than in EPI pigs fed formula only.Similarly, measured individual PUFAs, such as LA, ALA, and AA, weresignificantly higher in EPI pigs fed with formula pre-hydrolyzed withiRO than in EPI pigs fed with ND only. A trend increase in DHA freefatty acid concentration was also recorded.

FIG. 40A and FIG. 40B show the sum of polyunsaturated free fatty acidconcentration (mean±SD) in the healthy control group (n=6), EPI group(n=6), and EPI+iRO group (n=6) measured in pre-prandial blood samplesand post-prandial 1 h samples collected after 6 weeks of feeding of EPIpigs with ND or PND. Healthy pigs were fed with ND. There was astatistically significant p-value of *p<0.05 for the difference betweenthe groups using ordinary one-way ANOVA (p=0.0007 and p=0.0091, for thedifference between the groups for pre-prandial samples and 1 hpost-prandial samples, respectively).

As shown in FIG. 40A, FIG. 40B, and Table 15, post-prandial levels ofLC-PUFAs from the samples collected 1 h after a meal show increasedconcentration of free fatty acids in plasma collected from EPI pigs fedwith PND when compared to EPI pigs fed with ND. For example, thepost-prandial level of LA in EPI pigs fed with PND increased byapproximately 10% while the post-prandial level of LA in EPI pigs fedwith ND only increased by approximately 3%; the post-prandial level ofALA in EPI pigs fed with PND increased by approximately 35% while thepost-prandial level of LA in EPI pigs fed with ND only increased byapproximately 10%; and the post-prandial level of EPA in EPI pigs fedwith PND increased by approximately 3 folds while the post-prandiallevel of EPA in EPI pigs fed with ND only increased by approximately 1fold. This result again suggests enhanced absorption and effectivenessof the point-of-care approach. This result was encouraging, since theplasma concentrations of specific LC-PUFAs, such as LA, ALA, and EPA,were elevated 1 h after feeding with PND, which is usually the time whenplasma LC-PUFA levels begin elevating in the healthy pigs. Presumablydue to the complex hydrolysis and absorption process, mean LC-PUFAlevels were reaching maximal concentration in the plasma approximatelyabout 4 to about 6 hours after a meal.

TABLE 15 Comparison of the total and individual plasma free fatty acidPUFA concentration before and 1 h after a meal LA ALA AA EPA DHA Sum FAμg FA/ μg FA/ μg FA/ μg FA/ μg FA/ g FA/100 g 100 g 100 g 100 g 100 g100 g Groups sample sample Sample sample sample sample Healthy ON 0.27 ±0.04 59.0 ± 9.8* 1.9 ± 0.5* 35.0 ± 4.0* 0.7 ± 0.1 10.5 ± 1.2* (Control)fast 1 h  0.33 ± 0.07* 71.6 ± 17.1 3.2 ± 1.5* 37.0 ± 4.5* 1.7 ± 1   10.2± 1.2* EPI ON 0.16 ± 0.03 35.9 ± 7.7  1.0 ± 0.3  17.0 ± 4.0  0.7 ± 0.43.2 ± 0.7 fast 1 h 0.17 ± 0.02 37.1 ± 6.2  1.1 ± 0.3  18.0 ± 3.04 1.4 ±0.7 3.0 ± 0.6 EPI + ON 0.23 ± 0.07 47.6 ± 14.8 1.4 ± 0.5* 27.4 ± 12.50.7 ± 0.5 4.7 ± 2.2 iRO fast 1 h  0.26 ± 0.07* 52.4 ± 14.8 1.9 ± 0.6*26.3 ± 14   2.5 ± 1.3* 4.4 ± 2.2

Data shown in Table 15 is a sum of polyunsaturated free fatty acidconcentration (mean±SD) in healthy pigs (n=6, EPI n=6, and EPI+iRO n=6)for sum of all FA, but also LA, ALA, AA, EPA, and DHA measured inpre-prandial blood samples and post-prandial 1 h samples collected after6 weeks of feeding of EPI pigs with ND or PND. Healthy pigs were fedwith ND. The p-value is *p<0.05 for the difference between groups, ANOVApaired t-test.

Benefit of the consumption of PND was also demonstrated based on thegeneral increase of the total amount of free fatty acids in the plasmaof EPI+iRO pigs, whether on or off fast, compared to EPI pigs.

13.2.3.2 Tissue Accretion of LC-PUFA

Improved LC-PUFA absorption upon feeding with pre-hydrolized formula for6 weeks resulted in increased levels of AA and DHA in visceral tissue,as measured in the fat, liver, and heart, and neuronal tissue, asmeasured in the hypocampus. A summary of the results are shown in Table16.

TABLE 16 Selected LC-PUFA from fat, heart, liver, hippocampus from EPIand healthy pigs fed ND or PND for 6 weeks Groups p-Values Healthy EPIEPI + IRO Healthy FA (%) vs. Healthy EPI + IRO Tissues g FA/100 g FAEPI + IRO vs. EPI vs. EPI Fat AA 1.09 ± 0.05 0.46 ± 0.08 0.60 ± 0.18<0.001 <0.001 0.0121 DHA 0.82 ± 0.04 0.30 ± 0.05 0.46 ± 0.11 <0.001<0.001 <0.001 Σ Ω-3 2.38 ± 0.10 1.70 ± 0.28 1.90 ± 0.23 <0.001 <0.0010.0353 Σ Ω-6 14.34 ± 0.52  10.66 ± 1.70  12.04 ± 2.05  <0.001 <0.0010.0363 Heart AA 19.97 ± 3.25  15.62 ± 4.91  19.74 ± 3.08  0.43129 0.01030.0114 DHA 5.35 ± 1.06 2.44 ± 0.69 3.33 ± 1.33 <0.001 <0.001 0.0205 ΣΩ-3 7.18 ± 1.17 4.79 ± 1.12 5.24 ± 1.21 <0.001 <0.001 0.1661 Σ Ω-6 35.52± 6.95  36.93 ± 6.96  38.73 ± 6.15  0.11415 0.3119 0.2473 Liver AA 13.51± 1.11  4.33 ± 1.37 6.73 ± 4.90 <0.001 <0.001 0.0504 DHA 5.79 ± 0.311.25 ± 0.33 2.05 ± 1.26 <0.001 <0.001 0.0192 Σ Ω-3 7.19 ± 0.46 2.65 ±0.73 3.88 ± 1.20 <0.001 <0.001 0.0020 Σ Ω-6 26.59 ± 1.39  18.38 ± 2.38 22.29 ± 3.69  <0.001 <0.001 0.0018 Hippocampus AA 9.06 ± 1.05 7.94 ±1.02 8.68 ± 0.81 0.1097 0.0013 0.0090 DHA 8.33 ± 1.31 6.88 ± 1.27 7.65 ±0.84 0.0353 0.0009 0.0175 Σ Ω-3 9.44 ± 1.27 8.57 ± 1.36 8.82 ± 0.830.0430 0.0283 0.2559 Σ Ω-6 17.59 ± 1.47  16.78 ± 1.74  17.35 ± 2.09 0.3406 0.0706 0.1784

Data shown in Table 16 represents mean±SD levels of LC-PUFA from healthypigs (n=6, EPI n=6, and EPI+iRO n=7) collected from liver, fat, heart,and hippocampus tissue at the end of the study. EPI pigs were fed eitherwith ND or PND. Healthy pigs were fed with ND. The p-value is *p<0.05for the difference between groups, ANOVA paired t-test.

Improved absorption of LC-PUFA was demonstrated by reduced fecal fats,and increased concentration of total LC-PUFAs was reflected in liver,heart, and fat tissue accretion of AA and DHA. Lung tissue was alsoexamined, and no difference in AA levels was seen between groups(Healthy: 10.12±0.9, EPI: 10.07±1.4, and EPI+iRO: 10.17±1.33 g/100 g FA;p=NS); however, a slight increase in DHA levels in healthy pigs was seenwhen compared to EPI pigs (Healthy: 2.52±0.2, EPI: 1.68±0.2, andEPI+iRO: 1.72±0.4 g/100 g FA; p<0.05). For neuronal tissue, hippocampusand visual cortex were examined. As shown in Table 16 and FIG. 41,statistically significant positive changes were demonstrated for bothDHA and AA levels in the hippocampus.

Furthermore, the visual cortex was analyzed, and no difference betweenEPI pigs fed with ND or PND or healthy pigs was found (AA: Healthy:8.64±0.2, EPI: 8.74±0.4, and EPI+iRO: 8.45±0.24 g/100 g FA, p=NS; DHA:Healthy: 13.19±0.4, EPI: 12.76±10.52, and EPI+iRO: 12.32±0.8 g/100 g FA;p=NS for difference between EPI and EPI+iRO groups). This result is, tothat extent, in agreement with the work from C. Tyburczy et al.85:335-343 (2011), who looked at omega-3 and omega-6 changes inperipheral and central tissue in newborn pigs fed with milk replacersenriched with different amounts of TG-DHA and TG-AA during the first 28days of life. The sensitivity of different parts of the central nervoustissue to dietary DHA has been previously shown in numerous studies withterm and preterm neonatal non-human primates. The brain consistentlyshows increased region-specific DHA accretion related to the level andduration of performed DHA feeding. In our study, pigs were fed a formulaenriched with TG-DHA and TG-AA in the ratio of 2:1 (AA/DHA) for 6 weeks,which favored accretion of AA, which can explain why composition of DHAin the majority of the tested peripheral or central tissues wereincreased to a lesser extent when compared to AA levels. In addition, itis well known that the very same enzymes are involved in metabolism ofomega-6 and omega-3 PUFA and therefore different accumulation rates inplasma and tissues can be expected.

13.2.4 Enhanced Absorption of Vitamin A and Vitamin E

Improvement in the absorption in fat-soluble vitamins A and E was alsodemonstrated in the study (vitamin E: EPI 0.8±0.4 vs. EPI+iRO: 1.5±0.9,p<0.5; vitamin A: EPI: 0.18±0.06 vs. EPI+iRO: 0.26±0.17, p=NS). Most NDsare supplemented with vitamin A and vitamin E acetyl ester stable formsthat need to be digested by pancreatic carboxy ester hydrolase beforeabsorption. It is known that pre-term babies, newborn babies, kids, andadults with impaired pancreatic function have deficiency in thesefat-soluble vitamins. Thus, enhanced absorption of vitamin A and vitaminE in this study suggests that iRO can cleave respective acetyl esterforms and enhance their absorption (vitamin E: EPI 0.8±0.4 vs. EPI+iRO:1.5±0.9, p<0.5; vitamin A: EPI: 0.18±0.0.06 vs. EPI+iRO: 0.26±0.17,p=NS).

13.3 Summary

In summary, consumption of pre-hydrolyzed infant formula with iRO, i.e.,Rhizopus oryzae lipase attached to beads, was safe and led to improvedfat absorption, resulting in reduced total fat and LC-PUFA fat in thestool, reduced steatorrhea, normalized blood lipid profile, andincreased composition of LC-PUFA in cell membranes of heart, liver, fat,and hippocampus. Together, data from this nonclinical study suggeststhat consumption of a pre-hydrolyzed nutritional drink may be aneffective treatment for people with compromised pancreatic output notonly to simply increase caloric intake, but also to increase intake of“essential” free fatty acids, such as DHA and AA.

Example 14: 12-Day Efficacy Study of Exemplary Device 200 on EPI PigsFed Via G-Tube

This 12-day study tested the use of an exemplary device 200 duringnightly enteral (G-tube) feedings. Exemplary device 200 used in thisexperiment was substantially similar to that used in Example 3. Thestudy assessed the safety of device 200 during nightly G-tube feedingand whether prehydrolyzed fat enhances the absorption of total fat andlong-chain polyunsaturated fatty acids (omega-3) from completenutritional formula Peptamen AF® (Nestle Nutrition, EU). The efficacyand safety of device 200 in enteral feeding was tested in the porcinemodel of EPI disease, as described in Example 13.

This 12-day study was used to mimic the effects of device 200 fornightly supplemental G-tube feeding. Pancreatic duct ligation surgery,as described in Example 13, was performed on 14 pigs to create exocrinepancreatic inefficiency in this experiment. Out of the 14 operated pigs,only 11 pigs developed complete pancreatic insufficiency and were usedin this study.

Prior to pancreatic duct ligation surgery, following the surgery, andduring a pre-study period, pigs were orally fed a standard pig diet thatcontained 17.5% crude protein, 3.9% crude fiber, and 3.5% crude fat,5.2%, 5000 IE/kg vitamin A, 500 IE/kg vitamin D, and 85 mg/kg vitamin E.Feeding was done twice daily (2.0% body mass per meal) at 7 AM and 3 PM.

During the 12-day study, five EPI pigs in a control group were fed withnon-hydrolyzed Peptamen AF® via a G-tube, and six EPI pigs in a testgroup were fed Peptamen AF® pre-hydrolyzed using device 200 via aG-tube. Pigs were fed during the day with a standard solid feed similarto the mean human high-fat diet (about 1400 kcal/day/pig). In order tomimic nighttime enteral feeding, which would be a common use for device200 in EPI patients, the EPI pigs were supplemented with an additional750 calories (500 mL; 1.8 g Ω-3, Peptamen AF®, Nestlé Nutrition, EU)nightly at a flow rate of 2 mL/min over 4 hours via G-tube feeding.Device 200 used in the study was manually filled with 1 g of lipase 710attached to particles 300. Peptamen AF® is a semi-elemental enteralformula that provides pre-hydrolyzed protein. The use of PERT capsulesfor protein digestion was not provided, since Peptamen AF® containspre-hydrolyzed protein and the use of device 200 would efficientlyhydrolyze the fat. Pre-hydrolyzed proteins are stable in pre-packagedenteral formulas in contrast to free fatty acids and monoglycerides,which oxidize and quickly become rancid.

14.1 Study Design and Procedures

During this 12-day study period, EPI pigs were randomized into twogroups, control group (“PepAF”) and test group (“PepAF+Device”), basedon the body weight and health status, as shown in FIG. 42:

-   -   1) Control group: Five EPI pigs were enrolled and fed with solid        feed twice during the day at 7 AM and 3 PM. During the night        from 7 PM to 11 PM, 500 mL of non-hydrolyzed Peptamen AF® was        provided using G-tube feeding during the 4-hour period.    -   2) Test group: Six EPI pigs were enrolled and fed with solid        feed twice during the day at 7 AM and 3 PM. During the night        from 7 PM to 11 PM, 500 mL of Peptamen AF® formula        pre-hydrolyzed using device 200 was provided during the 4-hour        period of enteral feeding.

The experiment lasted 12 days, and the G-tube feeding was performedevery evening during the study. On the last 3 days of the study, three24 h stool and urine samples were collected. On the last day of thestudy, just before sacrificing, fasting morning blood samples werecollected for taking protein and fat profile and measurements of DHA andEPA levels in the blood as markers of LC-PUFA absorption.

Measurement of Fat and Protein Content in Food and Stool Samples

Stool samples were collected during the last 3 days (3×24 h) of the12-day study and weights were recorded. A small fraction from eachsample was measured for coefficient of protein absorption (% CPA) andtotal LC-PUFA.

Protein stool measurement was estimated based on the nitrogen fecallosses. Nitrogen levels were measured in food samples and in collectedfecal samples using a standard Kjedhal method. The coefficient ofprotein absorption (% CPA) was calculated as:

${C\; P\; A} = {\frac{\lbrack {{{nitrogen}\mspace{14mu} {intake}\mspace{11mu} ( \frac{g}{24\mspace{14mu} h} )} - {{nitrogen}\mspace{14mu} {in}\mspace{14mu} {feces}\mspace{14mu} ( {g\text{/}24\mspace{14mu} h} )}} \rbrack}{{nitrogen}\mspace{14mu} {intake}\mspace{14mu} ( {g\text{/}24\mspace{14mu} h} )} \times 100\%}$

Plasma lipid profile was estimated based on Lipaemic Index (LI).Lipaemic Index was calculated by:

Lipaemic Index=(OD 660 nm−OD 700 nm)×100%

Each plasma sample was measured in duplicate.

14.2 Results

All pigs had normal behavior, and no adverse events were recorded thatrelated to G-tube feeding through device 200. As shown in Table 17, foodconsumption was normal and similar between the EPI control group(“PepAF”) and test group (“PepAF+Device”) fed pre-hydrolyzed formula.Steatorrhea is a common symptom seen in people with compromisedpancreatic function (lipid malabsorption due to poor hydrolysis of fat)and was reduced in the test group when compared to the control group,shown by 72-hour stool weight.

There was a positive correlation between % CFA and plasma levels of EPA(rs=0.81; p=0.003), DHA (rs=0.672; p=0.027) and PUFA (r_(s)=0.736;p=0.013).

TABLE 17 Mean food intake and stool weight Groups Food intake (g) StoolWeight (g)* PepAF 388 ± 81  386.7 ± 77.3 PepAF + Device 363 ± 125 316.7± 97.7 *p = 0.014

One of the safety parameters considered important for this study wasgrowth. It should be noted that this was only a 12-day study usingnightly G-tube enteral feeding using device 200. Even with this shortduration using device 200, improved growth was observed in thePepAF+Device group (6.7% increase with test group vs. 5.3% increase forthe control group, p=NS). Body weight changes are shown in Table 18.

TABLE 18 Body weight change after 12 days of nighttime G-tube feeding BW(kg) Difference Group day 1 day 12 (kg) % Change Pep AF (n = 5) 15.7 ±0.9 16.5 ± 0.6 0.8 ± 0.7 5.3 ± 4.8 Pep AF + Device 15.2 ± 2.6 16.2 ± 2.61.0 ± 0.3 6.7 ± 2.2 (n = 6)

At the end of the study, blood samples were collected for estimation ofblood fat profile and levels of omega-3 fatty acids. As shown in Table19, plasma TG levels were normal and the same between the groups, butcholesterol and HDL were increased in the pigs fed pre-hydrolyzedPeptamen AF®, suggesting improved fat absorption and a trend towardsnormalization of cholesterol levels (normal cholesterol range in healthypigs is 3-4 mmol/L). The p-value is *p<0.05 for the difference betweenthe control and the PepAF+Device group in total cholesterol and HDLlevels. TG was within the normal range.

TABLE 19 Blood fat profile Group TG Cholesterol HDL LDL (n = 5-6)(mmol/L) (mmol/L) (mmol/L) (mmol/L) PepAF 0.59 ± 0.25 2.47 ± 0.15 0.92 ±0.15 1.17 ± 0.13 PepAF + 0.50 ± 0.22 2.81 ± 0.35* 1.27 ± 0.36* 1.25 ±0.09 Device

As a part of the safety tests, the morphometric structure (mucosalthickness and epithelial structure) of the small intestine after 12 daysof consecutive feeding with pre-hydrolyzed Peptamen AF® by device 200was observed and compared with the structure of pigs fed non-hydrolyzedPeptamen AF®. As a control, a group of healthy pigs and a group of EPIpigs fed the same solid high-fat diet feed (EPI pigs fed solid feedonly, no supplemental enteral G-tube feeding) were included.

The small intestine was chosen as one of the most vulnerable sites inthe GI and the part where most of the nutrients from food are absorbedinto circulation. In this study, the middle portion of the smallintestine was analyzed.

As shown in FIG. 43, results of the histopathological examination andmorphometry analysis of the samples from the small intestine demonstrateagain that consumption of pre-hydrolyzed formula by device 200 is safe,demonstrated by:

-   -   1) No pathological changes in the middle portion of the small        intestine independent of the use of pre-hydrolyzed or        non-hydrolyzed Peptamen AF®.    -   2) Slight trend increase in the mucosal thickness in        PepAF+Device group after only 12 days of pre-hydrolyzed G-tube        feeding when compared to control group fed non-hydrolyzed        formula.

Overall mucosal thickness was reduced in both EPI groups due to EPIdisease in pigs, independent of feeding with either pre-hydrolyzed ornon-hydrolyzed Peptamen AF® when compared to healthy pigs fed solidfeed. Interestingly, the mucosal thickness was improved in both PeptamenAF® G-tube fed groups when compared to EPI pigs fed only a solidhigh-fat diet, indicating remodelling capacity of the small intestine.

After only 12 days of nightly G-tube feeding, basal fatty acid DHA andEPA fasting blood levels in EPI pigs fed pre-hydrolyzed formula(PepAF+Device) increased to 727.6±164.9 ng/mL for DHA (p=0.008) and to512.6±81.6 ng/mL for EPA (p<0.001) when compared to the control group(PepAF) fed non-hydrolyzed formula, whose DHA level was 442.8±154.1ng/mL and EPA level was 190.8±23.1 ng/mL. FIG. 44 and Table 20 show themean change over time from baseline to day 12.

TABLE 20 Mean changes in DHA and EPA plasma levels after 12 days ofnightly feeding using device 200 in an exocrine pancreatic insufficiency(EPI) porcine model Group DHA (ng/mL) EPA (ng/mL) (n = 5-6) Baseline Day12 Change Baseline Day 12 Change PepAF + Device 214.2 ± 141.4 727.6 ±164.9 513.4* 43.3 ± 23.5 512.6 ± 81.6 469.3* PepAF 268.7 ± 129.2 442.8 ±154.1 174.1 81.5 ± 84.0 190.8 ± 23.1 109.3 Results are shown as a meanof group ± SD. DHA and EPA measured as ng/ml. *p = 0.008 for differencebetween Pep AF + Device vs. PepAF for DHA. **p = 0.001 for differencebetween Pep AF + Device vs. PepAF for EPA.

Results in Table 20 are shown as a mean of the group±SD. DHA and EPAwere measured as ng/ml. The p-value is *p=0.008 for the differencebetween Pep AF+Device vs. PepAF for DHA; **p=0.001 for differencebetween Pep AF+Device vs. PepAF for EPA.

The healthy control group had a mean baseline level of 753.3±102.2 ng/mLfor DHA and 138.1±10.0 ng/mL for EPA. This is indicative of theefficiency of device 200 to hydrolyze fats, including the most complexfats (longer carbon chains and double bonds), such as DHA and EPAtriglycerides, providing them in an easily absorbable form of free fattyacids and monoglycerides. Peptamen AF® has a total of 1.8 g of omega-3fat based on the label claim, primarily in the form of EPA and DHAtriglycerides. Since DHA and EPA levels are deficient in people withcystic fibrosis and developmental immature infants, this improvement inphysiologically relevant LC-PUFA fats in only 12 days of nightly G-tubefeeding using device 200 is an important finding with potentialbeneficial clinical implications.

In addition, total fatty acid changes in plasma were assessed at the endof 12 days of feeding for the test group fed with pre-hydrolyzed formulacompared to the control group fed with non-hydrolyzed formula. As shownin Table 21, increased uptake of specific long-chain polyunsaturatedfatty acids with the use of device 200 resulted in a statisticallysignificant reduction in the omega-6 to omega-3 ratio. The healthycontrol group had a mean baseline omega-6 to omega-3 ratio of 8.7±0.8.DHA and EPA measured as grams of DHA or EPA over 100 g of total fattyacids. The p-value is *p<0.05 for the difference between baseline andday 12.

TABLE 21 Change in omega-6 to omega-3 ratio after 12 days in an exocrinepancreatic insufficiency (EPI) porcine model Group (n = 5-6) BaselineDay 12 PepAF + Device 10.5 ± 0.7 2.4 ± 0.3* (pre-hydrolyzed) PepAF 10.6± 0.6 4.2 ± 0.6 (non-hydrolyzed)

To assess the effect of improved fat absorption and LC-PUFA absorption,bioavailability analysis of fatty acid content in the lung, retina,heart, liver, small intestine, and the erythrocytes (red blood cells(RBC)) of each pig was performed. Results of DHA and EPA accretion inthe respective tissues are shown in Table 22 and Table 23.

TABLE 22 DHA (g/100 g total fatty acids) Group Small Erythrocytes (n =5-6) Lung Retina Heart Liver Intestine (RBC) PepAF + Device 5.1 ± 0.3*9.6 ± 3.4* 1.8 ± 0.5 5.4 ± 0.3* 3.4 ± 0.3* 1.8 ± 0.3 PepAF 4.5 ± 0.5 8.2 ± 2.7  1.8 ± 0.5 4.7 ± 0.4  1.4 ± 0.1  1.8 ± 0.2

TABLE 23 EPA (g/100 g total fatty acids) Group Small Erythrocytes (n =5-6) Lung Retina Heart Liver Intestine (RBC) PepAF + Device 5.8 ± 0.5*1.2 ± 0.3* 1.7 ± 0.4* 6.2 ± 0.6* 3.3 ± 0.7*  1.2 ± 0.4* PepAF 3.3 ± 0.4 0.8 ± 0.4  1.3 ± 0.2  3.4 ± 0.3  2.4 ± 0.9  0.87 ± 0.1

Results are shown as a mean of group±SD, *p<0.05 for the differencebetween PepAF+Device vs. PepAF on day 12.

In all tested tissues, a significant increase in the levels of EPA wasdemonstrated in the test group fed with formula pre-hydrolyzed usingdevice 200 compared with the control group fed with non-hydrolyzedformula. Interestingly, even in RBCs that have a half-life of around 100days, a significant increase of 37% in EPA levels was observed. Measuredlevels of DHA were significantly elevated in all analyzed tissues withthe exception of the heart and RBCs.

Patients with compromised pancreatic output and/or fat malabsorptionhave a higher risk of fatty acid deficiencies in plasma and tissue,which may be related to a variety of adverse physiological effects, suchas altered membrane and cellular functions, as well reduced tolerabilityof formula due to poor hydrolysis of fats. Thus, enteral feeding usingdevice 200 may help in reducing such deficiencies and/or may normalizemucosal thickness, indicating a remodelling capacity of the smallintestine, as well as improving gastrointestinal symptoms.

To demonstrate changes in blood lipid profile after G-tube feeding,blood samples were collected on the last day of the study before solidmeals, 4 hours after the solid meals, and before and after enteralG-tube feeding. LI is a simple turbidometry method that is used tomeasure postprandial changes in total blood fat.

As shown in FIG. 45, total lipid absorption measured as a change in LIincreased in pigs from the test group fed with pre-hydrolyzed PeptamenAF® when compared to the control group fed with non-hydrolyzed PeptamenAF®. Calculated AUC_(t12-24h) values were significantly increased in thetest group fed with pre-hydrolyzed formula (11.1±1.29 vs. 20.8±8.6;p<0.05), indicating efficient delivery of easily absorbable fat whenusing device 200.

As shown in FIG. 46, a surprising result in this study was that proteinabsorption by the pigs of the test group fed with formula pre-hydrolyzedusing device 200 (PepAF+Device) improved by 9% compared with the controlgroup fed with non-hydrolyzed formula, as measured by changes in fecalnitrogen levels and expressed as a coefficient of protein absorption(61.2±0.9% vs. 66.9±2.8%, p=0.001). This is surprising, since PeptamenAF® already contains pre-hydrolyzed protein and no difference in proteinabsorption was expected. It is theoretically hypothesized thatpre-hydrolyzed fat from device 200 may lead to less un-hydrolyzed fat inthe GI tract, which may reduce inflammation, increase mucosal thickness,improve remodelling capacity of the small intestine, and thus maysupport enhanced absorption of protein and other nutrients.

As shown in Table 24, another surprising result in this study was thatuse of device 200 seemed to promote more efficient uptake of fat-solublevitamins (Vitamins D, E; p<0.05) for the test group. Fat-solublevitamins (A, D, E, K) have shown to be reduced in people withcompromised pancreatic output or fat malabsorption.

TABLE 24 Absorption of vitamins D and E Vitamin D Vitamin E Group (n =5-6) (ng/mL) (mcg/mL) PepAF + Device (hydrolyzed) 6.48 ± 2.78* 0.53 ±0.26* PepAF 3.82 ± 0.97 0.25 ± 0.07 (non-hydrolyzed) Normal range inhealthy pigs 5-20 1-8 *p < 0.05 for difference between baseline and day12

These unexpected results indicate that the test group fed with formulapre-hydrolyzed using device 200 may have better absorption of othernutrients in formula, such as proteins and vitamins, which eventuallymay be beneficial to the subject in need of the nutrients in theformula.

Example 15: Comparison of 24-Hour Pharmacodynamic Profiles of Total Fatand Free Fatty Acids in EPI Pigs after Single G-Tube Feeding UsingExemplary Device 200 and not Using any Device 200

This study is a pharmacodynamic proof of principle study performed toasses fat absorption from 500 mL Peptamen AF® (750 kcal, about 30%calories from 32 g TG-fat, Nestle Nutrition, EU) after a single G-tubefeeding using an exemplary device 200 when compared to standard G-tubefeeding. Exemplary device 200 used in this experiment was substantiallysimilar to that used in Example 3. The Peptamen AF® pre-hydrolyzed usingan exemplary device 200 (flow rate of 2 mL/min, test group, n=6) and thenon-hydrolyzed Peptamen AF® (control group, n=5) were administrated to“naïve” fasted EPI pigs over a period of about 5 hours. The EPI pigswere prepared as described in Example 13. After two days of wash outbefore the 24-hour treatment period, EPI pigs were returned to baselinelevels and then crossed over to the opposite group, and thus each pigserved as its own control. After the two days of wash out, a healthycontrol group of 3 pigs of the same age and breed were enrolled. Duringthis 24-hour test period, the only food provided to the pigs was viaG-tube.

The EPI pigs were randomized into the control group (“PepAF”) and thetest group (“PepAF+Device”) based on body weight and health status. Thecontrol group was fed with 500 mL of non-hydrolyzed Peptamen AF® viaG-tube, the test group was fed with 500 mL of Peptamen AF formulapre-hydrolyzed using device 200 via G-tube, and the healthy controlgroup of pigs was fed with non-hydrolyzed Peptamen AF® via G-tube,during the approximately 5-hour period. The G-tube feeding started atabout 10:00 AM, and blood samples were collected before the start ofG-tube feeding (basal collection) and at 1, 3, 5, 7, 10, 12, 16, 20, and24-hour time points.

Ten blood samples of each pig were collected over the 24-hour studyperiod for estimation of the total fat content in Lipaemic Index (LI).Also, changes in the concentrations of DHA and EPA free fatty acids weremeasured.

As shown in FIG. 47, fat absorption, as measured by lipaemic index (LI),was significantly improved in the PepAF+Device group when compared tothe PepAF group fed with non-hydrolyzed Peptamen AF® during and directlyafter the 5-hour feeding time. Calculated AUC_(0-10h) values weresignificantly increased in the PepAF+Device group when compared to thePepAF group (11.5±1.99 vs. 9.1±1.63; p=0.023), indicating improved fatabsorption with the use of device 200 in the enteral G-tube feedingcircuit.

Plasma concentrations of EPA and DHA upon G-tube feeding withpre-hydrolyzed PeptamenAF® were also measured, since these free fattyacids represent one of the most critical biomarkers of LC-PUFAabsorption. As shown in FIG. 48A and FIG. 48B, a significant improvementin the absorption of EPA and DHA fatty acid was demonstrated with theuse of device 200. Device 200 efficiently hydrolyzed EPA and DHA (themost complex and the longest triglyceride chains) for the test groupwhen compared to the control group. Importantly, the phramacodynamic24-hour profiles overlapped between healthy pigs and EPI pigs fed withpre-hydrolyzed formula via G-tube using device 200, indicating thatabsorption of PeptamenAF® pre-hydrolyzed using device 200 was almostnormalized compared to that of healthy pigs.

As shown in Table 25, formula hydrolyzed using device 200 was associatedwith a statistically significant increase in total fat absorption andimprovement in uptake of omega-3 fatty acids (DHA and EPA) in plasmalevels over 24 hours for the test group compared to the control groupfed with non-hydrolyzed formula (p<0.05).

TABLE 25 Changes in total DHA and EPA fatty acids over 24 hours in anexocrine pancreatic insufficiency (EPI) porcine model Group DHA EPA (n =5-6) Baseline 24-h Change Baseline 24-h Change Pep + Device 0.9 ± 0.22.1 ± 0.2 1.2* 1.0 ± 0.1 5.5 ± 0.7 4.5** PepAF 1.2 ± 0.2 1.6 ± 0.2 0.41.1 ± 0.2 2.1 ± 0.2 0.9 Healthy 1.6 ± 0.0 2.3 ± 0.2 0.7 1.2 ± 0.2 3.2 ±0.1 2 control

In Table 25, DHA and EPA are measured as grams of DHA or EPA over 100 gtotal fatty acids. Results are shown as a mean of the group±SD. Thep-values are as follows: *p=0.0005 for the difference betweenPepAF+Device vs. PepAF over 24 hours for DHA; **p<0.0001 for thedifference between PepAF+Device vs. PepAF over 24 hours for EPA.

As shown in Table 26, increased uptake of specific long-chainpolyunsaturated fatty acids with use of device 200 resulted in astatistically significant reduction in the omega-6 to omega-3 ratio.Previous studies have demonstrated that a balanced ratio of omega-6 toomega-3 fatty acids is beneficial in maintaining normal development,immunological function, and overall health.

TABLE 26 Change in omega-6 to omega-3 ratio over 24 hours in an exocrinepancreatic insufficiency (EPI) porcine model Group (n = 5-6) Baseline24-hours PepAF + Device 10.6 ± 0.4 3.6 ± 0.5* PepAF 10.5 ± 0.8 7.0 ± 1.2Healthy control  8.7 ± 0.8 5.2 ± 0.4

In Table 26, DHA and EPA are measured as grams of DHA or EPA over 100 gtotal fatty acids. Results are shown as a mean of the group±SD. Thep-value is *p<0.0001 for difference between baseline and 24 hours forPepAF+Device vs. PepAF.

The single delivery of formula pre-hydrolyzed using device 200 was safeand well tolerated with no vomiting or diarrhea recorded. G-tube feedingof 500 mL of Peptamen AF® pre-hydrolyzed using device 200 resulted insignificantly improved total fat absorption and a normalizedpharmacodynamic profile of physiologically relevant LC-PUFAs, such asEPA and DHA.

Example 16: Human Study of CF Patients to Evaluate Fat Absorption UsingDevice 200

A prospective, controlled, randomized, double-blind, cross-over study ofhuman patients with cystic fibrosis (CF) and compromised pancreaticoutput receiving enteral nutrition was performed to evaluate fatabsorption, GI symptoms, and tolerability of nutritional formula usingdevice 200. Device 200 used during this study is described in DeviceExample 1, below. Like patients with compromised pancreatic output,patients with CF have previously been shown to be deficient in LCPUFAs,including DHA and EPA. People with CF tend to have abnormal fatty acidmetabolism, with increased release and high turnover of AA and decreasedlevels of DHA, EPA, and LA in plasma, erythrocytes, platelets, andtissues.

Plasma measures generally allow for precise assessment of fatty acidabsorption, including from enteral feedings of nutritional formulas.Measuring plasma levels of DHA and EPA is believed to provide anaccurate assessment of DHA and EPA absorption by the body. Since only asmall amount (<1%) of DHA and EPA are internally synthesized, plasmalevels of DHA are primarily influenced by dietary intake. In addition,as 20- and 22-carbon-chain polyunsaturated fats, DHA and EPA are poorlyabsorbed relative to other fatty acids, such as simple medium-chainfatty acids and saturated fat. Therefore, changes in the plasma levelsof DHA and EPA after enteral feeding may be a sensitive indicator of fatabsorption and may serve as surrogate biomarkers representative of fatabsorption from diet in general. Using nutritional formulas containingfixed quantities of certain fatty acids, such as DHA and EPA, alsoallows for precise measurement of fat absorption following enteralfeeding. Accordingly, plasma levels of DHA and EPA were chosen asbiomarkers of fat in this study. While previous studies have looked atplasma uptake of fatty acids following ingestion of triglycerides, thisstudy looked at plasma uptake of fatty acids following ingestion ofpre-hydrolyzed triglycerides (i.e., free fatty acids and monoglycerides)generated using device 200.

Thirty-three patients with CF, ranging in age from 5 years to 34 years,were recruited as part of the study. The study comprised of a 7-daybaseline and run-in period (Period A), an 11-day double-blind crossoverperiod (Period B), and a 9-day open-label safety period (Period C). Eachpatient received two study treatments (device 200 or a placebo) in acrossover fashion during Period B.

During Period A (Days −7 to −1), baseline evaluations were performed onthe patients, enteral nutrition intake was standardized, and patientsmaintained a 7-day GI symptom diary, a tool developed to asses GIsymptoms associated with enteral nutrition administration. At studyentry, patients completed an Impact Questionnaire, a study-specific tooldeveloped to assess enteral nutrition use and practice, as well as toassess the impact of enteral nutrition on certain activities of dailyliving (ADLs). During this period, patients resumed their standard ofcare, including pancreatic enzyme replacement (PERT) use during the dayor with enteral feeding during the night.

On Day 1 of Period B (Days 1 to 11), patients were randomized in a 1:1ratio to a tube feeding session using Impact® Peptide 1.5 (Nestle HealthScience 750 kcal, 32 (g) fat and 2.45 (g) DHA/EPA per 500 mL) witheither an active device 200 or a placebo enteral device. The feedingsession lasted four hours. Patients returned on Day 9 for the second,crossover treatment. Patients who had received tube feeding with adevice 200 on Day 1 received a tube feeding with a placebo device on Day9, and vice versa. In this way, each patient acted as his or her owncontrol. The feeding session again lasted four hours. Days 1 and 9 wereseparated by a 7-day washout period. On administration Days 1 and 9,blood samples were collected to assess plasma fatty acid levels at hours0, 1, 3, 7, 9, 12, and 24. Plasma samples were analyzed forconcentrations of DHA and EPA using ultra high performance liquidchromatography (UHPLC).

During Period C (Days 12 to 20), all patients were instructed to usedevice 200 with standardized nocturnal enteral nutritional formula(Impact Peptide 1.5) from Days 12 to 18. Similar to Period A, patientsmaintained a GI symptom diary for 7 consecutive days and followed theirstandard of care. Repeat administration of the Impact Questionnaire wasperformed on the last day.

Study results indicated that use of device 200 improved tolerability toenteral feedings of nutritional formula and reduced GI symptoms whencompared to use of PERTs alone. Use of device 200 with up to 1,000 mL offormula decreased GI symptoms, and during Period C (use of device 200),both the incidence and severity of GI symptoms decreased compared toPeriod A. At the end of Period C, more patients reported an absence ofdigestive symptoms and reported that tube feeding did not decreaseappetite or ability to eat meals or snacks. Fewer patients skippedbreakfast when using device 200 compared to when using just PERTs (33%vs. 48.5%). This may be due to a reduction of GI symptoms (reducednausea, bloating, fullness), which allowed patients to feel hungry or tobe able to eat again. As a result, using device 200 may not onlyincrease caloric intake by increasing the amount of fats a patient'sbody may absorb, but also by allowing patients to eat more because theyhave fewer GI symptoms. The number of patients reporting individualsymptoms in Period A vs. Period C is shown below in Table 27. Onepatient did not complete the 7-day GI symptom diary in Period A.

TABLE 27 Number of patients reporting GI symptoms Period A Period CSymptom (n = 32)* (n = 32) Abdominal Pain 12 (38%)  9 (27%) Bloating 7(22%) 4 (12%) Constipation 6 (19%) 0 Diarrhea 7 (22%) 4 (12%) Gas 11(34%)  10 (30%)  Indigestion/Heartburn 7 (22%) 3 (9%)  Nausea 6 (19%) 4(12%) Steatorrhea 6 (19%) 3 (9%)  Vomiting 3 (9%)  3 (9%)  Other 0 2(6% )

Plasma levels of both DHA and EPA increased significantly during andafter administration of a single enteral tube feeding of 500 mL of anutritional formula using device 200. The maximum concentration of DHAand EPA in blood plasma occurred at the 7-hour time point and was nearly300% above baseline, as shown in FIG. 49A. Measurement ofbioavailability of DHA and EPA was determined by assessing the areaunder the curve (AUC₂₄), concentration peak (C_(max)) and time to maxconcentration (T_(max)) during the 24-hour interval period (TO to 24hours) for DHA and EPA (absolute and baseline adjusted). As shown inFIG. 49B, there was an absolute increase in total DHA and EPAconcentration in blood plasma. In fact, the increase in plasmaconcentration achieved using device 200 during this study brought theconcentrations of DHA and EPA within range of plasma levels generallyseen in normal populations (p<0.0001) for AUC₂₄.

Use of device 200 showed a statistically significant improvement inabsorption of both DHA and EPA (p<0.01), as well as LA (p<0.05). A 2.4fold improvement in total EPA and DHA absorption was observed, asmeasured by AUC₂₄, and there was a 2.2 fold improvement in total EPA andDHA absorption, as measured by C_(max). This improvement in EPA and DHAabsorption brought the fatty acid profiles of CF patients more in linewith the fatty acid profiles of the normal population.

Use of device 200 significantly increased LCPUFA absorption in apediatric sub-population (p<0.05). AUC for plasma concentrations of DHAand EPA were significantly higher with use of device 200 compared withplacebo. Similarly, the maximum plasma concentration in 24 hours(C_(max)) of DHA and EPA was significantly higher with use of device 200compared with placebo. Similar results were observed in all age groups,and the results were statistically significant in the child (5-12 yearsof age) and adolescent (13-21 years of age) study sub-populations, asshown in Table 28, below. Absolute changes seen between age groups inAUC may reflect a dose of DHA and EPA per kg of body weight.

TABLE 28 Mean (SD) AUC and C_(max) for plasma concentrations of DHA andEPA for study population and age group sub-populations, baselineadjusted All Ages 5-12 Ages 13-22 Ages 22+ (n = 33) (n = 14) (n = 16) (n= 3) AUC (ug/mL/h₀₋₂₄) Placebo 251.1 (163.6) 252.1 (100.4) 270.1 (212.7)144.7 (59.2)  Device 200 610.8 (307.6) 722.3 (402.8) 539.0 (191.6) 473.8(165.2) p <0.001 <0.001 <0.0027 NS C_(max) (ug/mL) Placebo 20.1 (13.6)22.2 (14.5) 18.6 (13.9) 11.6 (8.2) Device 200 42.8 (22.9) 48.1 (10.8)48.1 (10.8) 28.6 (7.0) p <0.001 <0.001 <0.001 NS

Patients who rely on nutritional formulas for a large portion of theirfood intake often have irregular fatty acid profiles. Their fat profilestend to show over-absorption of some fats, e.g., saturated fats andpalmitic acid, and show under-absorption of others, like LCPUFAs,particularly DHA, AA, and EPA. More complex fatty acids, includingLCPUFAs like DHA, AA, and EPA, are more difficult for the body to digestand subsequently absorb. The results from this study indicate that asfats become more complex (longer chain length and larger number ofdouble bonds), the magnitude of increase in absorption by the body—asindicated by increased plasma levels—increased with use of device 200.Less complex fats showed nominal increases in absorption with use ofdevice 200. This indicates that device 200 hydrolyzed the more-complexfats effectively (which are deficient in people with fat malabsorption,especially those with pancreatic immaturity or deficiency), allowing forincreased absorption of the more-complex LCPUFAs. The directrelationship between the complexity of the fat and the magnitude in theincrease in fat absorption may have helped change the fatty acidprofiles of CF patients in this study, making them look more like thefatty acid profiles of a normal population.

Since people with CF exhibit a deficiency in LCPUFAs, and since plasmauptake of LCPUFAs is slow, there is an initial physiological reductionfrom baseline for certain fats. Device 200 showed an ability to providereadily absorbable fatty acids, thereby reducing the baseline reductionseen when device 200 was not used.

Use of device 200 resulted in clinically meaningful increases inbioavailability of key physiologically relevant LCPUFAs (DHA, EPA) knownto be deficient in people with pancreatic immaturity and/or exocrinepancreatic insufficiency, like CF and fat malabsorption. The magnitudeof response in this study exceeded what would be expected in people withCF, since they are known to have not only a deficiency in uptake of DHAand EPA, but also a metabolic defect. However, the study indicates thatuse of device 200 to pre-hydrolyze LCPUFAs at the point of care allowedCF patients to more readily absorb total fats, but in particular,LCPUFAs, as indicated by increases in plasma content and in reduction ofGI symptoms, bringing the fatty acid profiles of the study patients morein line with those of a normal population. The ability to increaseLCPUFA uptake into plasma may play a role in inflammation levels in CFpatients. The ratio of AA to DHA is directly involved in maintaining aproper inflammatory response, and thus if device 200 is able to improvethe AA to DHA ratio, use of device 200 may also decrease CF symptoms,because pro-inflammatory products are responsible for increased mucusrelease and neutrophil influx and activation, resulting in additionalinflammation. Pro-inflammatory eicosanoid metabolites of AA(prostaglandins, leukotrienes, lipoxins) correlate with diseaseseverity.

By providing readily absorbable DHA and EPA using device 200, it may bepossible to more effectively outline a dose response expectation topromote more effective nutritional management.

Example 17: Evaluation of Device 200 Used to Administer Infant Formulain a Preterm Porcine Model

This study tested the use of device 200 during enteral g-tube feedingwith Similac Special Care 24 infant nutritional formula. Device 200 usedduring this study is described in Device Example 1, below.

The study assessed the safety, tolerance, and efficacy of device 200 forenteral feeding of pre-term piglets, an animal model that thatapproximates human babies born at approximately 30 weeks gestationalage. The study was intended to mimic enteral feeding in pre-term babies.The experiment was performed with 15 preterm piglets (8 male and 7female) delivered by Caesarean section from two sows at 7-8 days priorto full term (day 107/108; full term is 115 days). The study wasdesigned as a parallel 9-day efficacy study with 15 piglets randomizedbased on body weight and health status into two groups:

-   -   a. Group 1: control group with 7 piglets fed with non-hydrolyzed        Similac Special Care 24 with Iron (59 mL, 24 Kcal, 0.25% DHA and        0.40% AA, Abbott Nutrition); and    -   b. Group 2: treatment group with 8 piglets fed Similac Special        Care 24 with Iron after having been passed through device 200 to        pre-hydrolyze the fats.

Nutritional formula was delivered through device 200 at a flow rate of 1mL/min for the treatment group.

Similac Special Care 24 with Iron is representative of a typical pretermformula. It is an iron-fortified feeding formula for promoting growth inlow-birth-weight infants and premature infants. The fat content of theformula is a combination of medium chain triglycerides, soy, and coconutoils, and out of the total fat content, 0.25% is DHA and 0.40% is AA.

Results of the preterm study indicate that use of device 200 for enteralfeeding of pre-hydrolyzed fats for a period of 9 days was safe and welltolerated. The treatment group of piglets showed no adverse clinicalsigns, including no gastrointestinal intolerance, vomiting, diarrhea, orsigns of abdominal distension. Feeding volume was adjusted daily, basedon growth and feeding tolerance, and was similar between the two groupsduring the duration of the study (mean formula intake of 127 mL/kg/dayfor control group and 129 mL/kg/day for treatment group). The treatmentgroup also showed an overall increase in body weight, the development ofsuckling instinct, and growth of nails, hair, and muscle strength. Therewere also no histopathology findings in the small or large intestinethat could be attributed to the enteral feeding of pre-hydrolyzed fatsin the form of free fatty acids and monoglycerides.

Preterm infants often experience suboptimal growth, which may affectorgan development, vulnerability to infection, and respiratory orintestinal disorders. Suboptimal growth is generally a result of poorfat digestion and poor nutrient absorption due to immaturity of thepancreas and the intestinal tract, as well as lack ofbile-salt-stimulated lipase that is necessary for fat digestion andsubsequent fat absorption. Nine days of enteral feeding with nutritionalformula hydrolyzed using device 200 significantly improved fatabsorption, which resulted in an improved growth velocity of 3.6g/kg/day in the treatment group when compared to the control group(control group 17.5±6.6 vs. treatment group 21.1±4.6 g/kg/day). Indeed,a 21% increase in the growth velocity was recorded in the treatmentgroup when compared to the control group (p=0.179). It is important tonote that daily formula volumes were matched between groups.

To demonstrate the effect of the use of device 200 on blood plasmaconcentration levels of DHA and AA, blood levels were analyzed atbaseline (before use of device 200) and at the end of the treatmentperiod (9^(th) day of treatment). A significant increase of 15% and 22%,respectively, was shown in plasma DHA and AA concentrations in thetreatment group after 9 days of use. Increased plasma levels of DHA andAA from baseline through day 9 were as follows:

-   -   i. DHA:        -   1. Control: 51.6±7.4 to 51.4±15.8 ug/mL, p=NS        -   2. Treatment: 47.4±5.4 to 55.7±6.7 ug/mL, p=0.005    -   ii. AA:        -   1. Control: 95.4±16.5 to 105.3±32.1 ug/mL, p=NS        -   2. Treatment: 87.5±14.9 to 112.2±27.4 ug/mL, p=0.047

As shown above, DHA plasma levels at baseline in the control group were51.6±7.4 ug/mL and were unchanged at the end of the study (51.4±15.8ug/mL with a difference of 0.4 ug/mL). In contrast, in the treatmentgroup, DHA plasma levels increased by 8.3 ugL/mL from 47.4±5.4 ug/mL atbaseline to 55.7±6.7 ug/mL after 9 days of use (p=0.005).

Similar to the increase in concentration of DHA, plasma levels of AAincreased over the nine-day study with use of device 200 in thetreatment group by 24.7 ug/mL (a 28% increase) when compared to thecontrol group, which saw an increase of only 9.9 ug/mL (a 10% increase).

There were also significantly reduced fecal fat losses of criticalLCPUFAs in the treatment group as compared to the control group,suggesting improved fat absorption when piglets were fed pre-hydrolyzedfats using device 200. This reduction in fecal fat loss corresponds withthe improved plasma levels observed in the study. Levels of criticalfatty acids (g/100 g fatty acids of % total fat) in stool are shown inTable 29, below.

TABLE 29 Fecal LCPUFA content Omega- Omega- Groups PUFA LA AA DHA 6 3(g/100 g FA) Control 6.61 4.53 0.779 0.481 4.99 0.85 (3.26) (2.98)(0.248) (0.199) (3.47) (0.34) Treat- 2.79 2.06 0.178 0.142 2.29 0.32ment (1.71) (1.18) (0.172) (0.084) (1.27) (0.16) % Re- 58%↓ 54%↓ 77%↓70%↓ 54%↓ 63%↓ duction p value  0.011  0.038 0.001 0.002 0.045 0.003

Additionally, fecal content of medium chain fatty acids (C8-C12) wasalso lower by 54.7% with the use of device 200 (control 15.77±7.66 vs.treatment 7.13±4.96 g/100 g FA, p=0.009), indicating efficienthydrolysis of all triglycerides from infant formula.

There was also improved uptake of LCPUFAs into selected tissues, such asenterocytes of small intestine, in the treatment group compared to thecontrol group. Additionally, no negative impact on protein profile,glucose, triglycerides or cholesterol levels was observed in thetreatment group (there was no significant difference between thetreatment group and the control group, and levels were within normalrange for that age in both groups).

In summary, use of device 200 with g-tube enteral feeding with prematureinfant formula for 9 days was safe and well tolerated. The delivery ofpre-hydrolyzed fats resulted in improved body weight (in the targetedclinical range) and increased total and LCPUFA fat absorption, which wasdemonstrated by significantly increased plasma levels and a decrease infecal fat content.

Exemplary Devices 200 Device Example 1

An exemplary embodiment of device 200 may include a combination offeatures, as described below. Device 200 may include a hollow,cylindrical interior region, which may define a chamber 222. Chamber 222may have an interior diameter of approximately 1.56 cm, a height ofapproximately 1.94 cm, and a volume of approximately 3.70 mL. The outersurface of device body 210 may also be cylindrical, or may be shaped tofacilitate gripping. For example, an outer cross-section of device body210 may be polygonal, e.g., hexagonal. A length of device body 210 maybe approximately 4.42 cm, and a first connector 240 and a secondconnector 270 may extend from a top and a bottom region of device body210, respectively. The first and second connectors may be standard,ENFit connectors for use with enteral devices. First connector 240 maybe a female connector, and second connector 270 may be a male connector,or vice versa. The female connector may have an interior diameter at aninlet region that is larger than an interior diameter of the maleconnector at an outlet region, so that the female connector mayaccommodate the male connector within it. For example, first connector240 may have an interior diameter at an inlet region of approximately6.3 mm, or may otherwise be sized to meet the ENFit standard. Secondconnector 270 may have an interior diameter at an outlet region ofapproximately 1.9 mm, or may be otherwise sized to meet the ENFitstandard. The first and/or second connectors and device body 210 may beformed of a thermoplastic elastomer or rigid plastic, for example, apolycarbonate. In some embodiments, device body 210 may be made of arigid plastic, such as polycarbonate, which is transparent to allow auser to view the contents of device 200, e.g., particles 300 containedwithin chamber 222 or formula passing through device 200 during use.

An inlet filter 250 may be located adjacent inlet 212, and an outletfilter 260 may be located adjacent outlet 230 of device 200. The filtersmay both be tortuous path filters formed of polyethylene. As discussedabove, a tortious inlet filter 250 may promote dispersion of incomingnutritional formula more uniformly across chamber 222 or may promotedisruption of fat droplets and/or emulsification of the incomingnutritional formula. The outlet filter 270 may be a tortious filter inorder to effectively retain particles 300 within chamber 222. The inletand outlet filters may be the same type of filter in order to simplifymanufacturing or supply chain processes. The inlet filter diameter maybe approximately 15.0 mm, and inlet filter 250 may have a thickness ofapproximately 3.2 mm and a pore size of approximately 100 μm. The outletfilter diameter may be approximately 17.1 mm, and inlet filter 260 mayalso have a thickness of approximately 3.2 mm and a pore size ofapproximately 100 μm. In some embodiments, the specific sizes of theoutlet and inlet filters may depend in part of manufacturingconsiderations. For example, if press-fitting is used to incorporate thefilters into device 200, then the filter inserted first duringmanufacturing may be smaller in diameter than the filter inserted secondinto device 200.

Chamber 222 of this exemplary device 200 may contain particles 300 witha mean diameter of approximately 220 μm to approximately 350 μm with anormal particle size distribution, although alternative variations ofthis embodiment may include particles 300 with a mean diameter of up toabout 500 μm, for example, approximately 460 μm. Particles 300 may ormay not include fines (much smaller particles, e.g., having diameters ofless than approximately 50 um). Particles 300 may generally be sphericaland may have a mass density of approximately 0.25 g/mL to approximately0.36 g/mL and a particle moisture level of <5% when dry. Particles 300may be porous and may have pore diameters of approximately 10 nm toapproximately several hundred nm, which may be located on the surfaceand within the interior of individual particles 300. Particles 300 mayhave a mixture of smooth and textured surfaces. Particles 300 may beformed of approximately 58% ethylene glycol dimethacrylate, 41% A butylmethacrylate, and 1% glycidyl methacrylate. In alternative embodiments,particles 300 may be formed of approximately 60% ethylene glycoldimethacrylate, 39% butyl methacrylate, and 1% glycidyl methacrylate.Particles 300 may also include a functional group, e.g., approximately1% of an epoxy group (e.g., GMA). Exemplary variations of thisembodiment may contain epoxide levels (e.g., GMA) of approximately 0%,0.25%, 2%, or 5%. Particles 300 may also include approximately 7% to 10%of PEG, although, in some variations of this embodiment, less PEG or noPEG may be included on particles 300.

Particles 300 may include Rhizopus oryzae lipase immobilized primarilyby covalent binding. Approximately 50 mg to approximately 250 mg ofRhizopus oryzae lipase per gram of particle (by dry weight) may be boundto particles 300. In some embodiments, a highly purified Rhizopus oryzaelipase may be immobilized to particles 300 primarily by covalentbinding. The highly purified Rhizopus oryzae may have a greater abilityto hydrolyze fats as nutritional formula 110 is exposed to device 200.Approximately 5 mg to approximately 250 mg of purified Rhizopus oryzaelipase per gram of particle (by dry weight) may be bound to particles300.

Approximately 90-95% of chamber 222 may be filled with particles 300,leaving a headspace of approximately 5-10% of the chamber volume. Device200 may be filled by weight to achieve this headspace or may be filledaccording to volume. Depending on the particle density and size (whichmay vary slightly even from batch to batch of particles 300), averagefill weights for this embodiment may range from approximately 0.9 to 1.1g to approximately 1.0 to 1.2 g of particles loaded into chamber 222.This weight of particles 300 may be incorporated into chamber 222 toachieve a headspace of approximately 5-10% of chamber 222. In otherembodiments, chamber 222 may be filled with only reference to fillvolume, rather than fill weight.

Device 200 may be configured for use with a flow rate of fromapproximately 0.4 to 2.0 mL/min of nutritional formula 110 passingthrough device 200, and, in some embodiments, may be configured for usewith a flow rate of up to approximately 10.0 mL/min. The difference inflow rate between the flow rate set on pump 120 and the flow rateachieved through device 200 may be 10% or less. Device 200 may bedesigned for delivery of up to approximately 500 mL of nutritionalformula per feeding. Device 200 may be designed for delivery of up toapproximately 1,000 mL of nutritional formula per feeding. A device 200according to this embodiment may achieve more than 90% hydrolysisefficiency for most types of nutritional formulas.

Device Example 2

Exemplary embodiments of device 200 may also be configured toaccommodate faster flow rates of nutritional formulas passing throughthe device, e.g., to reduce feed time, or to accommodate greater volumesof nutritional formula per feed without compromising hydrolysisefficiency. For example, chamber 222 may have an increased heightcompared to Device Example 1, above, to accommodate more particles 300and/or more nutritional formula 110, and the length of the overall body210 may be taller to accommodate a taller chamber 222. For example, someembodiments may increase the height of chamber 222 by approximately 5 cm(for a total chamber height of approximately 6.94 cm) or byapproximately 2.91 cm (for a total chamber height of approximately 4.85cm), which may permit incorporation of up to approximately an additional3 g of particles 300 in chamber 222. In other embodiments, chamber 222may have similar dimensions to those of Device Example 1 or may have thesame dimensions.

Larger particle sizes may be used in devices 200 designed to accommodatea faster flow rate and/or larger quantity of nutritional formula 110being passed through device 200. For example, particles 300 may have anaverage diameter of approximately 375 μm or more with a normal particlesize distribution. Larger particles may reduce the likelihood ofobstruction or clogging that may be more likely to occur when higherflow rates, more viscous nutritional formulas, or larger volumes ofnutritional formulas are used. For example, some nutritional formulasmay produce semi-solid particles upon hydrolysis, which may collect indevice 200. If larger particles are used, then inlet and/or outletfilters with larger pore diameters, for example, approximately 100 μm toapproximately 150 μm, may also be used. Otherwise, device 200 of thisDevice Example 2 may be similar to device 200 of Device Example 1,above.

Device 200 of this example may accommodate use with flow rates of up toapproximately 10 mL/minute (600 mL/hour) or for use with volumes of upto 1,000 mL or more of nutritional formula per feed.

Device Example 3

Exemplary embodiments of device 200 may also be configured for use withpre-term babies, full-term babies, neonates, infants, and/or toddlers.For neonate or infant devices, for example, modifications may be made todevice 200 in some embodiments. For example, the volume of chamber 222may be reduced to approximately ½ to ¼ of the volume of the chamber ofthe device described in Device Example 1, above. Accordingly, thediameter and/or height of chamber 222 and of the overall device body maybe reduced to achieve this lower volume.

As described above, delivering nutritional formula 110 pre-hydrolyzedusing system 100 with device 200 may allow for direct delivery ofhydrolyzed and absorbable fatty acids to the GI tract of a subject priorto ingestion. Also, device 200 may be compatible with a wide range ofcomplex, commercially available nutritional formulas and may not affectnegatively other nutrients in the nutritional formula. Further, device200 may allow normalization of the calorie intake and fatty acid balanceand absorption of the subject, which may advantageously provide a morecontrolled option for healthcare providers to improve their managementand treatment of people with compromised pancreatic output or lipidmalabsorption.

In some embodiments, a method of supplying nutritional formula 110 usingdevice 200 may include the following steps. Step 1 may include preparinga source of nutritional formula 110. For example, step 1 may includeobtaining and/or preparing nutritional formula 110 of a predeterminedvolume in a container, e.g., a bag, vial, syringe, or bottle. Step 2 mayinclude fluidly connecting the source of nutritional formula 110 todevice 200 by using one or more tubes and connectors, such as first tube122 having tube connectors at its ends. Step 2 may further includeconnecting a first tube connector of first tube 122 to the source ofnutritional formula 110 and connecting a second tube connector of firsttube 122 to device 200 or first connector 240 of device 200. Step 3 mayinclude fluidly connecting device 200 to an enteral feeding tube. Forexample, step 3 may include connecting device 200 or second connector270 of device 200 to an enteral feeding tube or a connector to anenteral feeding tube. The enteral feeding tube may, for example, haveone end temporarily or permanently placed in fluid connection with theGI or nasogastric tract of a subject. Step 4 may include installing pump120 to system 100 and setting a flow rate of pump 120 for directingnutritional formula 110 through the tubes and device 200. Alternatively,pump 120 may be replace with a syringe. In the gravity feedingembodiments, step 4 may not be needed. Steps 1 to 4 may be performed inany order.

Step 5 may include directing nutritional formula 110 to device 200 usingpump 120, a syringe, or by the influence of gravity. Step 5 may furtherinclude priming nutritional formula 110 into and through device 200 andthe tubes, e.g., first tube 122 and enteral tube 124. Priming may beoperated automatically or manually by setting or adjusting pump 120 tofill device 200 and the tubes with nutritional formula 110 before thetubes are connected to the patient. Priming system 100 may reduce theamount of air dispensed into the patient prior to feeding of nutritionalformula 110. Pump 120 may operate at a faster speed during priming thanduring enteral feeding of nutritional formula 110. In such embodiments,device 200 may be designed to ensure that the faster pump rates thatoccur during priming do not damage or alter the operation of device 200.Step 5 may also include flushing, which may be performed automaticallyor manually. For example, in reusable embodiments, a pump may be set toa flush mode to purge a solution through the pump tubing to adequatelyvoid any residue formula, allowing the tubing to be used more than once.

Step 6 may include directing nutritional formula 110 through inlet 212,inlet filter 250, and particles 300 in chamber 222 of device 200. Insome embodiments, step 6 may further include distributing nutritionalformula 110 through inlet filter 250 and across particles 300 in chamber222. Step 7 may include allowing lipase 710 on particles 300 of device200 to be exposed to and/or to interact with the fat molecules innutritional formula 110 by directing and/or distributing the flow ofnutritional formula 110 across particles 300. Step 7 may further includeallowing particles 300 to mix with nutritional formula 110 and to movewith the flow dynamics of nutritional formula 110. Step 7 may alsoinclude allowing lipase 710 on particles 300 to hydrolyze thetriglycerides having LC-PUFAs in nutritional formula 110. In someembodiments, steps 6 and 7 may happen at substantially the same time.

Step 8 may include directing nutritional formula 110 through outletfilter 260 and outlet 282 while retaining particles 300 in device 200.Step 9 may include directing nutritional formula 110 through the enteralfeeding tube to the patient. Step 10 may include disconnecting device200 from system 100, disposing of device 200 and/or particles 300,and/or sterilizing and drying device 200.

In alternative embodiments, multiple devices 200 may be connected toeach other in series (tandem) or in parallel. When nutritional formula110 is flowed through device 200, fats contained in nutritional formula110 contact the surfaces of particles 300, and the fats may behydrolyzed from triglyceride form into free fatty acids andmonoglycerides via interaction with the lipase on particles 300. Theextent of fat hydrolysis may be determined in part by the contact (orresidence) time of the formula with particles 300 within chamber 222, aswell as the cumulative number of particles 300 to which nutritionalformula 110 is exposed. Increasing either the residence time or thenumber of particles to which nutritional formula 110 is exposed mayyield greater fat hydrolysis. Therefore, in cases in which a singledevice 200 does not alone provide a desired hydrolysis efficiency, thetandem arrangement of two devices 200 may increase hydrolysis, forexample, when used with certain nutritional formulas 110.

Connecting multiple devices 200 in series (tandem) may effectivelyincrease the cumulative residence time and the total number of particlesto which nutritional formula 110 is exposed. Arranging multiple devices200 in tandem may, for example, be useful when hydrolyzing largervolumes of nutritional formula 110 or when hydrolyzing nutritionalformula 110 at faster rates. To connect multiple devices 200 in series,second connector 270 of a first device 200 may be inserted directly intofirst connector 240 of a second device 200, or second connector 270 of afirst device 200 may connect to tubing, which may then connect to afirst connector 240 of a second device 200. The tandem devices may beconnected to the source of nutritional formula, tubing, and the patient,and used in a similar manner as described in steps 1-10 above.

A preliminary test assessed the effects of connecting multiple devices200 in series versus the use of a single device. In the preliminarytest, 1,000 mL of Peptamen® was flowed through a single device 200 at arate of 2 mL/minute, and 1,000 mL of Peptamen® was flowed through twodevices 200 connected In tandem at a rate of 2 mL/minute. The mean %hydrolysis of fats in the resulting hydrolyzed nutritional formula was92% for the single device 200 and 98% for the tandem setup. The testresults indicate that the tandem configuration may achieve hydrolysisefficiencies that are as high or higher than the hydrolysis efficienciesachieved by a single device 200.

Alternatively, rather than connecting multiple devices in series, thesame formula may be flowed through a single device 200 more than once toeffectively increase the total residence time and particle exposure. Forexample, a device 200 may be connected to a first end of an ‘empty’feeding circuit that is not yet attached to a patient. The second end ofthe ‘empty’ feeding circuit may be connected to a source of nutritionalformula. The ‘empty’ circuit may then be loaded with nutritional formula110 by drawing nutritional formula 110 up from the source and throughdevice 200 to a reservoir, which would expose the nutritional formula toone pass through device 200. The circuit would then be disconnected fromthe source of nutritional formula and instead attached to a patient. Thefeeding would then proceed as usual, i.e., nutritional formula 110 wouldbe flowed from the reservoir, through chamber 222 of device 200, and tothe patient. This would constitute a second pass through device 200.

The impact of this double-pass method on hydrolysis was assessed inpreliminary testing comparing a single-pass (regular) method of usingdevice 200 to the double-pass method of using device 200. In the test,two devices 200 were filled with a smaller quantity of particles 300(375 mg), and 50 mL of Similac® Special Care® 24 Cal infant formula waspassed through the devices at a flow rate of 2 mL/minute. For the firstdevice, 50 mL of the formula was passed through once (single-pass). Forthe second device, 50 mL of the formula was passed through twice(double-pass). To simulate a single-pass method, a syringe was loadedwith formula, a device 200 was attached to the syringe, and thenutritional formula was flowed from the syringe through the device. Tosimulate a double-pass method, a device 200 was attached to an emptysyringe. The nutritional formula was then drawn through the device toload the syringe, and then the nutritional formula was flowed out of thesyringe and through the device. The percentage of hydrolyzed fats in theformula flowed through using the single-pass method and the formulaflowed through using the double-pass method was then measured.

The measured % hydrolysis of the single-pass method was 37%, and themeasured % hydrolysis of the double-pass method was 63% in thispreliminary trial. The preliminary test data indicates that amultiple-pass method may increase the % hydrolysis of nutritionalformulas. Multiple-pass methods may be used for patients generally ormay be used in scenarios in which there is a limitation on the totalnumber of particles that may be used each day by a patient, or alimitation on the total number of particles that may be used at the sametime. For example, regulatory restrictions may limit the total amount ofparticles 300 to which a patient may be exposed in a single day. Thereduction in particles 300 used per feeding may, however, lowerhydrolysis efficiency of device 200. This reduction in hydrolysisefficiency may be offset, or at least partially offset, by using amulti-pass method to boost % hydrolysis by increasing residence timeand/or exposures to the particles 300. Thus, multiple-pass methods maybe useful during food preparation, for example, to increase % hydrolysiswithout introducing significant additional steps or changes to themethod of using device 200, particularly for infant nutritional formulapreparation in a NICU.

The many features and advantages of the present disclosure are apparentfrom the detailed specification, and thus, it is intended by theappended claims to cover all such features and advantages of the presentdisclosure that fall within the true spirit and scope of the presentdisclosure. Further, since numerous modifications and variations willreadily occur to those skilled in the art, it is not desired to limitthe present disclosure to the exact construction and operationillustrated and described, and accordingly, all suitable modificationsand equivalents may be resorted to, falling within the scope of thepresent disclosure.

Moreover, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be used as a basis fordesigning other structures, methods, and systems for carrying out theseveral purposes of the present disclosure. Accordingly, the claims arenot to be considered as limited by the foregoing description.

We claim:
 1. An enteral feeding device for hydrolyzing triglycerides ina nutritional formula by exposing the nutritional formula to lipase, thedevice comprising: a body housing a chamber; an inlet configured tofluidly couple with a source of nutritional formula, allowing thenutritional formula to enter the device from the source and flow intothe chamber; an outlet configured to fluidly couple with an enteralfeeding tube, allowing the nutritional formula to exit the chamber andflow into the enteral feeding tube; a plurality of particles containedwithin the chamber, wherein the lipase is covalently bonded to theplurality of particles; a headspace contained within the chamberdefining a space not occupied by the plurality of particles; an inletfilter located between the inlet and the chamber, wherein the inletfilter contains a first plurality of openings; and an outlet filterlocated between the chamber and the outlet, wherein the outlet filterhas a second plurality of openings, and wherein the second plurality ofopenings are smaller than the plurality of particles; wherein thetriglycerides in the nutritional formula are hydrolyzed as they passthrough the plurality of particles contained within the chamber.
 2. Thedevice of claim 1, wherein the plurality of particles, when dry, fill atleast 50% of the chamber.
 3. The device of claim 1, wherein theplurality of particles, when dry, fill at least 80% of the chamber. 4.The device of claim 1, wherein the plurality of particles, when dry,fill at least 90% of the chamber.
 5. The device of claim 1, wherein theplurality of particles, when exposed to the nutritional formula, fill atleast 80% of the chamber.
 6. The device of claim 1, wherein theplurality of particles, when exposed to the nutritional formula, fill atleast 90% of the chamber.
 7. The device of claim 1, wherein theplurality of particles swell so that, when dry, the plurality ofparticles fill less of the chamber than when exposed to the nutritionalformula.
 8. The device of claim 1, wherein an outside surface of atleast one of the plurality of particles is at least partiallyhydrophobic.
 9. The device of claim 1, wherein at least one of theplurality of particles is formed of one or more of ethylene glycoldimethacrylate, butyl methacrylate, or glycidyl methacrylate.
 10. Thedevice of claim 9, wherein at least one of the plurality of particles isformed of between about 50% to about 60% of ethylene glycoldimethacrylate by weight, between about 30% to about 45% of butylmethacrylate by weight, and between about 0.01% to about 10% of glycidylmethacrylate by weight.
 11. The device of claim 1, wherein at least oneof the plurality of particles is formed of between about 0% to about 10%of polyethylene glycol by weight.
 12. The device of claim 1, wherein atleast one of the plurality of particles has a substantially solidcross-section.
 13. The device of claim 1, wherein at least one of theplurality of particles has a substantially smooth outer surface.
 14. Thedevice of claim 1, wherein at least one of the plurality of particleshas a textured outer surface.
 15. The device of claim 1, wherein atleast one of the plurality of particles has a porous cross-sectionforming internal surfaces within the at least one particle.
 16. Thedevice of claim 15, wherein a median or a mean diameter of a pore of theporous cross-section ranges from about 1 nm to about 50 μm.
 17. Thedevice of claim 15, wherein the lipase is covalently bonded to theinternal surfaces.
 18. The device of claim 1, wherein at least one of anouter surface or an internal surface of at least one of the plurality ofparticles includes a functional group.
 19. The device of claim 18,wherein the functional group is an epoxy group, and the lipase iscovalently bonded to the epoxy group.
 20. The device of claim 1, whereina median or a mean diameter of the plurality of particles is betweenabout 100 μm and about 800 μm.
 21. The device of claim 1, wherein theplurality of particles comprises a first group of particles and a secondgroup of particles, wherein the first group of particles has a median ora mean diameter of that is different than a median or a mean diameter ofthe second group of particles.
 22. The device of claim 1, wherein anamount of the lipase covalently bonded to the plurality of particlesfalls within a range of about 5 mg to about 500 mg of lipase per 1 g ofthe plurality of particles.
 23. The device of claim 1, wherein at leastone of the first plurality of openings or the second plurality ofopenings includes a plurality of tortuous paths.
 24. The device of claim1, wherein the inlet filter is coated with at least one emulsifierconfigured to emulsify the nutritional formula as it passes through theinlet filter.
 25. The device of claim 1, wherein the inlet filter andthe outlet filter each have a thickness of between about 0.1 mm to about10 mm.
 26. An enteral feeding device for hydrolyzing triglycerides in anutritional formula by exposing the nutritional formula to lipase, thedevice comprising: a body housing a chamber, the chamber comprising: aplurality of particles contained within the chamber, wherein the lipaseis covalently bonded to the plurality of particles; and a headspacecontained within the chamber defining a space not occupied by theplurality of particles; a first connector configured to fluidly couplethe device with a first tube; an inlet positioned between the firstconnector and the chamber and fluidly coupled with the first connectorand the chamber; a second connector configured to fluidly couple thedevice with a second tube; an outlet positioned between the secondconnector and the chamber and fluidly coupled with the chamber and thesecond connector; and an outlet filter located between the chamber andthe outlet, wherein the outlet filter has a plurality of openings, andwherein the plurality of openings are smaller than the plurality ofparticles; wherein the triglycerides in the nutritional formula arehydrolyzed as they pass through the plurality of particles containedwithin the chamber.